Sterol carrier protein‐2:Not just for cholesterol any more

Although sterol carrier protein-2 (SCP-2) mediates cholesterol esterification in L-cell fibroblasts and stimulates an accumulation of cholesterol in these cells, a potential role for SCP-2 in fatty acid uptake and trafficking has not been appreciated. Certainly, recent experiments have shown that SCP-2 binds fatty acids in vitro with an affinity similar to that observed for fatty acid binding proteins. Because of the ubiquitous tissue distribution of SCP-2, as opposed to the specific distribution of fatty acid binding proteins, as well as the need for fatty acid trafficking in all cells, I have recently proposed that SCP-2 is the universal fatty acid trafficking protein. This supposition is based on a number of observations made with L-cell fibroblasts expressing either the 13.2 kDa SCP-2 or the 15 kDa proSCP-2. In L-cells expressing the 13.2 kDa SCP-2, fluorescent fatty acid uptake was increased by 10–30% depending upon the probe used. In 15 kDa proSCP-2 expressing cells, fluorescent fatty acid uptake was increased 20–40% depending upon the probe used. However, only expression of the 15 kDa pro-SCP-2 increased the cytoplasmic diffusion of the fluorescent fatty acid. Expression of either protein increased the uptake of [³H]-oleic acid 1.9-fold compared to control, with targeting of [³H]-oleic acid for esterification into cholesteryl esters. The 13.2 kDa SCP-2 did target a significant amount of [³H]-oleic acid for esterification into the triacylglycerol pool. Expression of either protein markedly reduced total cellular phospholipid levels, however both proteins increased cholesteryl ester levels. Interestingly, expression of the 15 kDa proSCP-2 decreased ethanolamine plasmalogen levels with a concomitant increase in choline plasmalogen. Expression of both proteins increased PUFA content of the phospholipids, although this effect was greater in 15 kDa proSCP-2 expressing cells. Hence, expression of SCP-2 increased fatty acid uptake and targeted fatty acid to unique lipid pools, suggesting that SCP-2 may effectively serve as universal fatty acid binding and trafficking protein.     

High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization

Canine myocardial sarcolemma was purified, and its phospholipid constituents were determined by gas chromatography-mass spectrometry, fast atom bombardment mass spectrometry, and conventional techniques. Canine myocardial sarcolemma contained 2.7 mumol of lipid Pi/mg of protein which was comprised predominantly of choline glycerophospholipids (47%), ethanolamine glycerophospholipids (28%), and sphingomyelin (11%). Sarcolemmal phospholipids contained 40% plasmalogen which was quantitatively accounted for by choline (57% of choline glycerophospholipid) and ethanolamine (64% of ethanolamine glycerophospholipid) plasmalogens. Choline plasmalogens contained predominantly the vinyl ether of palmitic aldehyde though ethanolamine plasmalogens were composed predominantly of the vinyl ethers of stearic and oleic aldehydes. The majority of sarcolemmal ethanolamine glycerophospholipids (75%) contained arachidonic acid esterified to the sn-2 carbon. Sphingomyelin was composed predominantly of long-chain saturated fatty acids (stearic and arachidic) as well as substantial amounts (8%) of odd chain length saturated fatty acids. The possible functional role of these unusual phospholipid constituents is discussed.     

Heart fatty acid uptake is decreased in heart fatty acid-binding protein gene-ablated mice

Cell culture systems have demonstrated a role for cytoplasmic fatty acid-binding proteins (FABP) in lipid metabolism, although a similar function in intact animals is unknown. We addressed this issue using heart fatty acid-binding protein (H-FABP) gene-ablated mice. H-FABP gene ablation reduced total heart fatty acid uptake 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6 compared with controls, respectively. Similarly, the amount of fatty acid found in the aqueous fraction was reduced 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6, respectively. Less [1-(14)C]16:0 entered the triacylglycerol pool, with significant redistribution of fatty acid between the triacylglycerol pool and the total phospholipid pool. Less [1-(14)C]20:4n-6 entered each lipid pool measured, but these changes did not alter the distribution of tracer among these pools. In gene-ablated mice, significantly more [1-(14)C]16:0 was targeted to choline and ethanolamine glycerophospholipids, whereas more [1-(14)C]20:4n-6 was targeted to the phosphatidylinositol (PtdIns) pool. H-FABP gene ablation significantly increased PtdIns mass 1.4-fold but reduced PtdIns 20:4n-6 mass 30%. Consistent with a reported effect of FABP on plasmalogen mass, ethanolamine plasmalogen mass was reduced 30% in gene-ablated mice. Further, 20:4n-6 mass was reduced in each of the three other major phospholipid classes, suggesting H-FABP has a role in maintaining steady-state 20:4n-6 mass in heart. In summary, H-FABP was important for heart fatty acid uptake and targeting of fatty acids to specific heart lipid pools as well as for maintenance of phospholipid pool mass and acyl chain composition.     

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.     

Brain lipid metabolism in the cPLA2 knockout mouse

We examined brain phospholipid metabolism in mice in which the cytosolic phospholipase A(2) (cPLA(2,) Type IV, 85 kDa) was knocked out (cPLA(2)(-/-) mice). Compared with controls, these mice demonstrated altered brain concentrations of several phospholipids, reduced esterified linoleate, arachidonate, and docosahexaenoate in choline glycerophospholipid, and reduced esterified arachidonate in phosphatidylinositol. Unanesthetized cPLA(2)(-/-) mice had reduced rates of incorporation of unlabeled arachidonate from plasma and from the brain arachidonoyl-CoA pool into ethanolamine glycerophospholipid and choline glycerophospholipid, but elevated rates into phosphatidylinositol. These differences corresponded to altered turnover and metabolic loss of esterified brain arachidonate. These results suggests that cPLA(2) is necessary to maintain normal brain concentrations of phospholipids and of their esterified polyunsaturated fatty acids. Reduced esterified arachidonate and docosahexaenoate may account for the resistance of the cPLA(2)(-/-) mouse to middle cerebral artery occlusion, and should influence membrane fluidity, neuroinflammation, signal transduction, and other brain processes.     

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids – phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd₂Gro) – and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd₂Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd₂Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd₂Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids – Ptd₂Gro >> PtdSer > PtdIns – underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes – PtdCho and PtdEtn – was detected. MS‐studies revealed the presence of hydroxy‐, hydroperoxy‐ as well as hydroxy‐/hydroperoxy‐species of Ptd₂Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd₂Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H₂O₂, confirmed that molecular identities of the products formed were similar to the ones generated during STS‐induced neuronal apoptosis. The temporal sequence of biomarkers of STS‐induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd₂Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd₂Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.     

Identification of plasmalogen as the major phospholipid constituent of cardiac sarcoplasmic reticulum

The phospholipid molecular species of canine myocardial sarcoplasmic reticulum were identified by fast atom bombardment mass spectrometry, reverse-phase high-performance liquid chromatography, and other conventional techniques. Cardiac sarcoplasmic reticulum contains 1.4 mumol of lipid Pi/mg of protein which is comprised of 53% plasmalogen. Cardiac sarcoplasmic reticulum ethanolamine glycerophospholipid contains 73% plasmalogen that is predominantly comprised of moieties with 18-carbon vinyl ethers at the sn-1 position and arachidonic acid at the sn-2 position. In contrast, canine skeletal muscle sarcoplasmic reticulum contains only 19% plasmalogen that is predominantly comprised of ethanolamine plasmalogen (78% of skeletal muscle sarcoplasmic reticulum ethanolamine glycerophospholipid) with arachidonic and docosatetraenoic acids at the sn-2 position. The possibility that tetraenoic ethanolamine plasmalogens in both cardiac and skeletal muscle sarcoplasmic reticulum facilitate calcium translocation by their propensity for adopting a hexagonal II conformation at physiologic temperatures is discussed.     

Supplemental feeding of phospholipid-enriched alkyl phospholipid from krill relieves spontaneous atopic dermatitis and strengthens skin intercellular lipid barriers in NC/Nga mice

Plasmalogen (Pls) is a glycerophospholipid derived from alkyl phospholipid (Alk) with antioxidant functions in vivo. The present study investigated the effects of ether phospholipids, such as Pls and Alk, on intercellular lipid barriers in the skin of NC/Nga mice, a model of atopic dermatitis (AD). NC/Nga mice fed Alk showed increased plasma levels of Alk and Pls. The AD-related changes in ceramide composition in the skin were abrogated by oral administration of Alk. Moreover, Alk suppressed skin inflammation in AD mice. These results indicate that Alk partially fortifies the stratum corneum lipid barrier and may be an effective treatment for AD.

Abbreviations: Pls: plasmalogen; PlsCho: choline plasmalogen; PlsEtn: ethanolamine plasmalogen; Alk: alkyl phospholipid; TJ: tight junction; FA: fatty acid; AD: atopic dermatitis; SO: soybean oil; FO: fish oil; DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; TG: triglyceride; PL: phospholipid; RF: retention factor; AlkCho: choline-type alkyl phospholipid; AlkEtn: ethanolamine-type alkyl phospholipid; LC-MS/MS: liquid chromatography-tandem mass spectrometry; FAR1: fatty acyl-coenzyme (Co)A reductase 1     

Effects of Maturation on the Phospholipid and Phospholipid Fatty Acid Compositions in Primary Rat Cortical Astrocyte Cell Cultures

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.     

Lipids from nerve tissues of the horseshoe crab, Limulus polyphemus.

1. The predominant lipids of nerve cords, ganglion and brain from horseshoe crabs were cholesterol (11% of lipid) and phospholipid (81% of lipid). 2. Major phospholipids were phosphatidyl ethanolamine and phosphatidyl choline with lesser amounts of phosphatidyl serine and phosphatidyl inositol and sphingomyelin. 3. The phospholipid fraction was characterized by a high content of plasmalogen, i.e. alk-1-enyl acyl phosphatides, so that 42% of the ethanolamine phosphatides were the plasmalogen, phosphatidal ethanolamine. 4. Phosphatidyl choline and phosphatidyl ethanolamine were high in polyunsaturation with 20:4 and 20:5 major fatty acids. Sphingomyelin had predominantly long chain saturated fatty acids. 5. Cerebrosides and gangliosides, which are associated with vertebrate nerve tissues, were absent from nerves of horseshoe crabs.     

Effect of cryopreservation on lipids and some physiological features of spermatozoa from rams pastured in highlands and in valleys

The effect of low temperature preservation on the motility and morphology of acrosomes, acrosomal proteolytic activity, phospholipid and fatty acid composition of phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE), and the cholesterol/phospholipid molar ratio in sperm from rams housed in the highlands or in the valleys, were studied. The indices of motility and morphological integrity of sperm from highland rams were much greater compared with those of valley rams. Phosphatidyl choline (PC) of the highland rams was more unsaturated, while PE was more saturated compared with those of valley rams. Cryopreservation of the sperm from highland rams significantly increased the content of choline plasmalogen, accompanied by a slight rise in the levels of lysophosphatidyl choline (LPC) and phosphatidyl inositol (PI) in their sperm. The fatty acid composition altered following cryopreservation. These variations were mainly due to a decrease in the amount of docosahexaenic acid and an increase in the amounts of linoleic and palmitic fatty acids. The results may be indicative of the fact that the alterations in the sperm of the valley rams were more pronounced and they may be attributed to the structural features of the sperm, as well as a reduced concentration of oxygen in the organs and tissues of the highland rams.     

Composition and fatty acid content of rat ventral prostate phospholipids

The major phospholipids of rat ventral prostate have been separated and examined using thin-layer chromatography, gas chromatography and mass spectrometry. The main phospholipid classes were choline and ethanolamine glycerophospholipids, accounting for 77.9% of total lipid phosphorus. The prostate also contained small amounts of serine glycerophospholipids and sphingomyelin. The relative proportions of fatty acids in the different phospholipid classes were also determined. Arachidonic acid in prostatic phospholipids is contributed primarily by ethanolamine glycerophospholipids. This fraction contained 65-69 mol% plasmalogens, whereas choline and serine glycerophospholipid fractions contained less than 5 mol% plasmalogens. Ethanolamine, choline and serine plasmalogens contained mainly vinyl ethers of palmitic and stearic aldehydes. Ethanolamine plasmalogens also contained the vinyl ether of oleic aldehyde.     

EFFECT OF 1-AMINOCYCLOPENTANE-1-CARBOXYLIC ACID (CYCLOLEUCINE) ON DEVELOPING RAT CENTRAL NERVOUS SYSTEM PHOSPHOLIPIDS

Treatment of developing rats with 1-amino-cyclopentane carboxylic acid (cycloleucine) resulted in changes in brain and spinal cord phospholipid content and fatty acid composition. General findings were a decrease in ethanolamine phospholipid content, and relative increase in the saturated fatty acid content of ethanolamine phospholipid. In all the different cycloleucine experiments conducted, there was consistently less fatty aldehyde present in the methylated ethanolamine phospholipid fatty acid-fatty aldehyde fractions than in corresponding controls. In some experiments fatty aldehyde was almost completely absent, suggesting the presence of little plasmalogen. Changes in fatty acids of phosphatidyl choline, the other phospholipid examined in this manner, were generally minor. Administration of massive amounts of sodium propionate in addition to cycloleucine did not result in an appreciable odd-chain fatty acid increase in the CNS. Examination of the spinal cords by electron microscopy demonstrated considerable myelin splitting in one set of animals. No other ultrastructural changes were evident. The suitability of this drug to produce a neurological condition and pathological state similar to that seen in B₁₂-deficient subacute combined degeneration is discussed.     

Phospholipids and phospholipid-bound fatty acids and aldehydes of spermatozoa and seminal plasma of rhesus monkeys.

The major components of the phospholipids of rhesus monkey spermatozoa are phosphatidyl choline (33%), phosphatidyl ethanolamine (25%), ethanolamine plasmalogen (16-1%), sphingomyelin (8-1%), choline plasmalogen (6-9%) and cardiolipin (4-5%). The major phospholipid-bound fatty acids are 16:0, 18:0, 18:1 and 22:6; the major fatty aldehydes are 15:0, 16:0 and 18:2. The same phospholipids are also present in the seminal plasma.     

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

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

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

Influence of dietary n-3 fatty acids on macrophage glycerophospholipid molecular species and peptidoleukotriene synthesis

The study examined the ability of dietary n-3 fatty acids to modify mouse peritoneal macrophage glycerophospholipid molecular species and peptidoleukotriene synthesis. After a 2-week feeding period, fish versus corn oil feeding significantly (P less than 0.01) lowered n-6 polyunsaturated fatty acid (PUFA) mol % levels, i.e., arachidonic acid (20:4n-6) in diacylphosphatidylserine (PtdSer), diacylphosphatidylinositol (PtdIns), diacylglycerophosphoethanolamine (PtdEtn), alkenylacylglycerophosphoethanolamine (PlsEtn), and diacylglycerophosphocholine (PtdCho). A notable exception was alkylacylglycerophosphocholine (PakCho), where only moderate decreases in 16:0-20:4n-6 and 18:0-20:4n-6 species were observed after fish oil supplementation. The predominant n-3 PUFA in macrophage phospholipid subclasses was docosapentaenoic acid (22:5n-3). The major n-3 species were 18:0-22:5n-3 in PtdIns, PtdSer, glycerophosphoethanolamines (EtnGpl) and 16:0-22:5n-3 in PtdCho and PlsEtn. The major n-3-containing species in PakCho were 16:0-20:5n-3 and 18:1-22:6n-3. These findings indicate that n-3 PUFA are differentially incorporated into macrophage phospholipid subclasses after dietary fish oil supplementation, and suggest that phospholipid remodeling enzymes selectively discriminate between substrates based on compatibility of sn-1 covalent linkage and the composition of the sn-1 and sn-2 aliphatic chains. Macrophage peptidoleukotriene synthesis was also strongly influenced after fish oil feeding; the LTC5/LTC4 ratio was significantly higher (P less than 0.01) in fish oil-fed animals than in corn oil-fed animals, 0.85 versus 0.01, respectively. These ratios were subsequently compared to phospholipid molecular species 20:5n-3/20:4n-6 ratios in order to determine potential sources of eicosanoid precursors.