Specific Phospholipid Classes Research Articles

T-cell senescence induced by peripheral phospholipids.

We present an integrated analysis of the clinical measurements, immune cells, and plasma lipidomics of 2000 individuals representing different age stages. In the study, we explore the interplay of systemic lipids metabolism and circulating immune cells through in-depth analysis of immune cell phenotype and function in peripheral dynamic lipids environment. The population makeup of circulation lymphocytes and lipid metabolites changes dynamically with age. We identified a major shift between young group and middle age group, at which point elevated, immune response is accompanied by the elevation of specific classes of peripheral phospholipids. We tested the effects in mouse model and found that 10-month-dietary added phospholipids induced T-cell senescence. However, the chronic malignant disease, the crosstalk between systemic metabolism and immunity, is completely changed. In cancer patients, the unusual plasma cholesteryl esters emerged, and free fatty acids decreased. The study reveals how immune cell classes and peripheral metabolism coordinate during age acceleration and suggests immune senescence is not isolated, and thus, system effect is the critical point for cell- and function-specific immune-metabolic targeting. • The study identifies a major shift of immune phenotype between young group and middle age group, and the immune response is accompanied by the elevation of specific classes of peripheral phospholipids; • The study suggests potential implications for translational studies such as using metabolic drug to regulate immune activity.

Lipid Dys-Homeostasis Contributes to APOE4-Associated AD Pathology.

The association of the APOE4 (vs. APOE3) isoform with an increased risk of Alzheimer's disease (AD) is unequivocal, but the underlying mechanisms remain incompletely elucidated. A prevailing hypothesis incriminates the impaired ability of APOE4 to clear neurotoxic amyloid-β peptides (Aβ) from the brain as the main mechanism linking the apolipoprotein isoform to disease etiology. The APOE protein mediates lipid transport both within the brain and from the brain to the periphery, suggesting that lipids may be potential co-factors in APOE4-associated physiopathology. The present study reveals several changes in the pathways of lipid homeostasis in the brains of mice expressing the human APOE4 vs. APOE3 isoform. Carriers of APOE4 had altered cholesterol turnover, an imbalance in the ratio of specific classes of phospholipids, lower levels of phosphatidylethanolamines bearing polyunsaturated fatty acids and an overall elevation in levels of monounsaturated fatty acids. These modifications in lipid homeostasis were related to increased production of Aβ peptides as well as augmented levels of tau and phosphorylated tau in primary neuronal cultures. This suite of APOE4-associated anomalies in lipid homeostasis and neurotoxic protein levels may be related to the accrued risk for AD in APOE4 carriers and provides novel insights into potential strategies for therapeutic intervention.

Patterns of mitochondrial membrane remodeling parallel functional adaptations to thermal stress.

The effect of temperature on mitochondrial performance is thought to be partly due to its effect on mitochondrial membranes. Numerous studies have shown that thermal acclimation and adaptation can alter the amount of inner-mitochondrial membrane (IMM), but little is known about the capacity of organisms to modulate mitochondrial membrane composition. Using northern and southern subspecies of Atlantic killifish (Fundulus heteroclitus) that are locally adapted to different environmental temperatures, we assessed whether thermal acclimation altered liver mitochondrial respiratory capacity or the composition and amount of IMM. We measured changes in phospholipid headgroups and headgroup-specific fatty acid (FA) remodeling, and used respirometry to assess mitochondrial respiratory capacity. Acclimation to 5°C and 33°C altered mitochondrial respiratory capacity in both subspecies. Northern F. heteroclitus exhibited greater mitochondrial respiratory capacity across acclimation temperatures, consistent with previously observed subspecies differences in whole-organism aerobic metabolism. Mitochondrial phospholipids were altered following thermal acclimation, and the direction of these changes was largely consistent between subspecies. These effects were primarily driven by remodeling of specific phospholipid classes and were associated with shifts in metabolic phenotypes. There were also differences in membrane composition between subspecies that were driven largely by differences in phospholipid classes. Changes in respiratory capacity between subspecies and with acclimation were largely but not completely accounted for by alterations in the amount of IMM. Taken together, these results support a role for changes in liver mitochondrial function in the ectothermic response to thermal stress during both acclimation and adaptation, and implicate lipid remodeling as a mechanism contributing to these changes.

Dietary fatty acid composition and the homeostatic regulation of mitochondrial phospholipid classes in red muscle of rainbow trout (Oncorhynchus mykiss).

Although dietary lipid quality markedly affects fatty acid (FA) composition of mitochondrial membranes from rainbow trout red muscle (Oncorhynchus mykiss), mitochondrial processes are relatively unchanged. As certain classes of phospholipids interact more intimately with membrane proteins than others, we examined whether specific phospholipid classes from these muscle mitochondria were more affected by dietary FA composition than others. To test this hypothesis, we fed trout with two diets differing only in their FA composition: Diet 1 had higher levels of 18:1n-9 and 18:2n-6 than Diet 2, while 22:6n-3 and 22:5n-6 were virtually absent from Diet 1 and high in Diet 2. After 5 months, trout fed Diet 2 had higher proportions of phosphatidylcholine (PC) and less phosphatidylethanolamine (PE) in mitochondrial membranes than those fed Diet 1. The FA composition of PC, PE and cardiolipin (CL) showed clear evidence of regulated incorporation of dietary FA. For trout fed Diet 2, 22:6n-3 was the most abundant FA in PC, PE and CL. The n-6 FA were consistently higher in all phospholipid classes of trout fed Diet 1, with shorter n-6 FA being favoured in CL than in PC and PE. Despite these marked changes in individual FA levels with diet, general characteristics such as total polyunsaturated FA, total monounsaturated FA and total saturated FA were conserved in PE and CL, confirming differential regulation of the FA composition of PC, PE and CL. The regulated changes of phospholipid classes presumably maintain critical membrane characteristics despite varying nutritional quality. We postulate that these changes aim to protect mitochondrial function.

Phospholipid makeup of the breast adipose tissue is impacted by obesity and mammary cancer in the mouse: Results of a pilot study.

Obesity, an established risk factor for breast cancer (BC), is associated with systemic inflammation. The breast contains adipose tissue (bAT), yet whether it plays a role in BC progression in obese females is being intensively studied. There is scarce knowledge on the lipid composition of bAT in health and disease. The purpose of this pilot study was: 1) to determine whether obesity and BC are associated with inflammatory changes in bAT 2) to analyze for the first time the lipid profile of bAT in obese and lean mammary tumor-bearing and normal mice. Syngeneic E0771 mammary tumor cells were implanted into the mammary fat pad of lean and diet-induced obese C57BL/6 mice. BATs were analyzed four weeks after tumor cell inoculation by immunohistochemistry and mass spectrometry. Phospholipids were identified and subjected to ratiometric quantification using a TSQ Quantum Access Max triple quadrupole mass spectrometer utilizing precursor ion scan or neutral ion loss scan employing appropriate class specific lipid standards in a two step quantification process. Four main classes of phospholipids were analyzed: phosphatidylcholines phosphatidylserines, phosphatidylethanolamines and phosphatidylinositols. Our results showed that bAT in obese (normal and tumor-bearing) mice contained hypertrophic adipocytes compared with their corresponding samples in lean mice; higher numbers of macrophages and crown-like structures were observed in obese tumor bearers compared to obese normal mice. BAT from normal obese mice revealed higher concentrations of phosphatidylethanolamines. Furthermore, bAT from tumor-bearing mice expressed higher phosphatidylcholines than that from non-tumor bearing mice, suggesting the presence of the tumor is associated with phosphatidylcholines. Conversion of phosphatidylethanolamines to phosphatidylcholines will be investigated in E0771 cells. Additional studies are projected to investigate macrophage activation by these specific classes of phospholipids. Occurrence of triglycerides and free fatty acids will be examined in bAT and similar lipidomic analyses will be carried out visceral adipose tissue, highly inflamed in obesity.

Identification of unusual phospholipid fatty acyl compositions of Acanthamoeba castellanii.

Acanthamoeba are opportunistic protozoan pathogens that may lead to sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba followed by aggressive treatment regimen. The plasma membrane of Acanthamoeba consists of 25% phospholipids (PL). The presence of C20 and, recently reported, 28- and 30-carbon fatty acyl residues is characteristic of amoeba PL. A detailed knowledge about this unusual PL composition could help to differentiate Acanthamoeba from other parasites, e.g. bacteria and develop more efficient treatment strategies. Therefore, the detailed PL composition of Acanthamoeba castellanii was investigated by 31P nuclear magnetic resonance spectroscopy, thin-layer chromatography, gas chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry. Normal and reversed phase liquid chromatography coupled with mass spectrometric detection was used for detailed characterization of the fatty acyl composition of each detected PL. The most abundant fatty acyl residues in each PL class were octadecanoyl (18∶0), octadecenoyl (18∶1 Δ9) and hexadecanoyl (16∶0). However, some selected PLs contained also very long fatty acyl chains: the presence of 28- and 30-carbon fatty acyl residues was confirmed in phosphatidylethanolamine (PE), phosphatidylserine, phosphatidic acid and cardiolipin. The majority of these fatty acyl residues were also identified in PE that resulted in the following composition: 28∶1/20∶2, 30∶2/18∶1, 28∶0/20∶2, 30∶2/20∶4 and 30∶3/20∶3. The PL of amoebae are significantly different in comparison to other cells: we describe here for the first time unusual, very long chain fatty acids with Δ5-unsaturation (30∶35,21,24) and 30∶221,24 localized exclusively in specific phospholipid classes of A. castellanii protozoa that could serve as specific biomarkers for the presence of these microorganisms.

Localization of anionic phospholipids in Escherichia coli cells.

Cardiolipin (CL) is an anionic phospholipid with a characteristically large curvature and is of growing interest for two primary reasons: (i) it binds to and regulates many peripheral membrane proteins in bacteria and mitochondria, and (ii) it is distributed asymmetrically in rod-shaped cells and is concentrated at the poles and division septum. Despite the growing number of studies of CL, its function in bacteria remains unknown. 10-N-Nonyl acridine orange (NAO) is widely used to image CL in bacteria and mitochondria, as its interaction with CL is reported to produce a characteristic red-shifted fluorescence emission. Using a suite of biophysical techniques, we quantitatively studied the interaction of NAO with anionic phospholipids under physiologically relevant conditions. We found that NAO is promiscuous in its binding and has photophysical properties that are largely insensitive to the structure of diverse anionic phospholipids to which it binds. Being unable to rely solely on NAO to characterize the localization of CL in Escherichia coli cells, we instead used quantitative fluorescence microscopy, mass spectrometry, and mutants deficient in specific classes of anionic phospholipids. We found CL and phosphatidylglycerol (PG) concentrated in the polar regions of E. coli cell membranes; depletion of CL by genetic approaches increased the concentration of PG at the poles. Previous studies suggested that some CL-binding proteins also have a high affinity for PG and display a pattern of cellular localization that is not influenced by depletion of CL. Framed within the context of these previous experiments, our results suggest that PG may play an essential role in bacterial physiology by maintaining the anionic character of polar membranes.

Differential distribution of DHA-phospholipids in rat brain after feeding: A lipidomic approach

On a per-weight basis, the brain is the organ richest in lipids, including a remarkable proportion of polyunsaturated fatty acids (PUFAs) of the omega 3 series, namely eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. The cerebral effects of exogenous DHA likely depend on its degree of incorporation into neuronal phospholipids and on its distribution among the various brain structures, after intake. Hence, because PUFAs are not evenly distributed among the brain phospholipid classes and because the existence of class-specific phospholipases that regulate their turnover, we sought to investigate the incorporation of omega 3 PUFAs in selected brain areas regions and specific phospholipid classes. Rats ( n=7) were administered, by oral gavage, 100 mg/kg/d of a commercially available fish oil (containing ∼84% of long-chain omega 3 fatty acids, of which ∼38% of DHA and ∼46% of EPA). Control rats ( n=7) received liquid paraffin. This treatment was continued for 30 days. Thereafter, we dissected three areas, namely the hippocampus, the striatum, and the cortex. Quantization of individual phospholipid classes and their molecular species was performed by ESI-MS/MS. Principal component analysis was used to examine the variation of the molecular lipid profiles (as percentage) induced by omega 3 supplementation. Our results show that provision of omega 3 fatty acids to rats results in their incorporation into brain phospholipids, the extent of which is lower in the striatum as compared with cortex and hippocampus. These data might in part explain the mixed therapeutic results obtained in neurological disorders, many of which are likely region-specific.

Myocardial Lipidomics. Developments in myocardial nuclear lipidomics

The development of electrospray ionization mass spectrometry has been critical for the analyses of lipidomes from subcellular organelles. The myocardial nuclear lipidome likely has a key role in the molecular regulation of gene expression. In fact, recent studies have suggested that specific phospholipid classes bind and regulate specific transcription factors. The dynamic regulation of the myocardial nuclear lipidome may be critical in mediating long-term pathological responses to stresses such as ischemia, tachycardia, and hypertension. In this brief review, the preparation of myocardial nuclei is discussed, and the resulting nuclear lipidome from rat and rabbit are shown as examples. The rabbit myocardial nuclear lipidome contains relatively more plasmenylcholine/phosphatidylcholine molecular species in comparison to that ratio observed in the rat myocardial nuclear lipidome. The composition of the rat myocardial nuclear choline glycerophospholipid pool was relatively enriched with molecular species containing arachidonic acid and docosahexaenoic acid in comparison to that in the rabbit myocardial nuclear choline glycerophospholipid pool. While the ethanolamine glycerophospholipids of the rabbit myocardial nuclei are enriched with arachidonic acid and plasmalogens, the ethanolamine glycerophospholipid profile from rat myocardial nuclei show less plasmalogen and more species containing docosahexaenoic acid. Last, significant differences in the ethanolamine glycerophospholipid molecular species were observed in the rabbit heart lipidomes from the nucleus and the mitochondria. Quantitation of these lipid species in hearts subjected to pathophysiological stresses may provide important information on the role of the myocardial nuclear lipidome on long-term cardiac cell function.

Insulin‐status‐dependent modulation of FoF1‐ATPase activity in rat liver mitochondria

Early and late effects of alloxan diabetes and insulin treatment on mitochondrial membrane structure and function were evaluated by studying the kinetic properties of mitochondrial membrane marker enzyme FoF1-ATPase and its modulation by membrane lipid/phospholipid composition and membrane fluidity. Under all experimental conditions the enzyme displayed three kinetically distinguishable components. In 1 wk-old diabetic animals the enzyme activity was unchanged; however, K(m) and V(max) of component I increased and K(m) of component II decreased. Insulin treatment resulted in lowering of K(m) and V(max) of components II and Ill. One-mon diabetic state resulted in decreased enzyme activity, whereas insulin treatment caused hyperstimulation. K(m) of components I and II decreased together with decreased V(max) of all the components. Insulin treatment restored the K(m) and V(max) values. In late-stage diabetes the catalytic efficiency of components I and II increased; insulin treatment had drastic adverse effect. Binding pattern of ATP was unchanged under all experimental conditions. Diabetic state resulted in progressive decrease in energy of activation in the low temperature range (E(L)). Insulin treatment lowered the energy of activation in the high temperature range (E(H)) without correcting the E(L) values. The phase transition temperatures increased in diabetic state and were not corrected by insulin treatment. Long-term diabetes lowered the total phospholipid content and elevated the cholesterol content; insulin treatment had partial restorative effect. The membrane fluidity decreased in general in diabetic condition and was not corrected by insulin treatment at late stage. Regression analysis studies suggest that specific phospholipid classes and/or their ratios may play a role in modulation of the enzyme activity.

Selective Peroxidation and Externalization of Phosphatidylserine in Normal Human Epidermal Keratinocytes During Oxidative Stress Induced by Cumene Hydroperoxide

Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar oxidative stress in vivo would under go phosphatidylserine oxidation/translocation. This would make them targets for macrophage recognition and phagocytosis, and thus limit their potential to invoke inflammation or give rise to neoplastic transformations.

14] Peroxidation of phosphatidylserine in mechanisms of apoptotic signaling

Oxidative stress is a frequent trigger of apoptosis in a variety of cells and is also thought to be involved as a component of the common pathway in the execution of apoptosis. While effects of oxidative stress on protein components of apoptotic machinery, such as caspases, have been well characterized, information on selective oxidation of specific classes of phospholipids in live cells is limited. The direct measurement of oxidized lipid products is problematic, as cells possess a very effective system for remodeling and repairing oxidatively modified phospholipids that interferes with their accurate measurement. The authors have developed a sensitive, specific, and reliable procedure for the assessment of oxidative stress in different classes of membrane phospholipids in intact live cells based on their metabolic acylation with an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (cis-PnA), as a reporter molecule. Basic procedures and some applications of the technique are described in the chapter. The model of cis-PnA-labeled cells offers a unique model system for quantitative studies of oxidative stress and selective oxidation of specific classes of phospholipids under normal physiological conditions, as well as during cell injury and apoptotic death.

Lipids and fatty acids of two pelagic cottoid fishes ( Comephorus spp.) endemic to Lake Baikal

Matured females of two Lake Baikal endemic fish species, Comephorus baicalensis and Comephorus dybowski, have been investigated for lipid of the whole body and specific tissues (liver, muscles, ovaries), phospholipid classes and fatty acids of neutral and polar lipids. Total lipid in the body (38.9% fresh weight), liver (23.5%) and muscles (14.5%) of C. baicalensis were greater than those of C. dybowski (4.7, 8.7 and 2.6%, respectively); only their ovaries were similar (5.3 and 5.6% lipid, respectively). In both species, phosphatidylcholine and phosphatidylethanolamine were the major phospholipids, ranging from 60.7 to 75.1% of total phospholipid and 14.5–25.7%, respectively. In most cases, monounsaturated fatty acids (MUFA) were the major fatty acid group in C. baicalensis, whereas polyunsaturated fatty acids (PUFA) were the major group in C. dybowski. The MUFA 18:1(n-9) prevailed over other fatty acids in C. baicalensis and varied from 19% in polar lipids of muscles to 56.1% in neutral lipids of muscles. In polar lipid of C. dybowski, the PUFA 22:6(n-3) prevailed over other fatty acids in muscles and ovaries, while 16:0 dominated polar liver lipids and neutral lipids of all tissues. Other major fatty acids included 16:1(n-7), 18:1(n-7), and 20:5(n-3). Values of the (n-3)/(n-6) fatty acid ratio for neutral lipids of C. baicalensis (0.5–0.9) are well below the range of values characteristic either for marine or freshwater fish, while these values for polar lipids (1.6–1.8) are in the range typical of freshwater fish. Neutral lipid fatty acid ratios in C. dybowski (2.5–3.1) allow it to be assigned to freshwater fish, but polar lipids (2.8–3.7) leave it intermediary between freshwater and marine fish.

Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain

Comamonas testosteroni P15 and its mutant strain E23 can tolerate and utilize phenol as the sole source of carbon and energy at up to 15 mM and 20 mM, respectively. Compared to the wild type P15, mutant E23 showed higher values of Ks and Ki but a lower μmax value, and had lower phenol hydroxylase and catechol 2,3-dioxygenase activities. Without phenol exposure, mutant E23 demonstrated a two-fold greater amount of cardiolipin than the wild type P15. Upon exposure to phenol, an increase in cardiolipin at the expense of phosphatidylethanolamine was observed in the wild type P15. However, there was no significant difference in major phospholipid contents between mutant E23 cells grown in the presence or absence of phenol. It was noted that the ratio of trans/cis fatty acids of phosphatidylethanolamine and cardiolipin in mutant E23 was 65–70% higher than that in the wild type P15. In the absence of phenol, the degree of saturation of cardiolipin in mutant E23 was 33% higher than that in wild type P15. In contrast to earlier findings, an increase in C16:1 9trans with a simultaneous decrease in C18:1 11cis instead of C16:1 9cis was observed in specific classes of phospholipids.

Random versus selective membrane phospholipid oxidation in apoptosis: role of phosphatidylserine.

The formation of reactive oxygen species has been associated with apoptosis. To assess the role of lipid peroxidation in apoptosis, we used 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN) to generate peroxyl radicals within cellular membranes of HL-60 cells. cis-Parinaric acid (cis-PnA) metabolically integrated into phospholipids of HL-60 cells was used as a probe to assess the extent of lipid peroxidation within specific phospholipid classes. Within 2 h, AMVN (500 microM) randomly oxidized more than 85% of cis-PnA contained in all major classes of phospholipids. AMVN-induced lipid peroxidation was followed by apoptosis as determined by nuclear condensation, DNA fragmentation, and annexin V binding to externalized phosphatidylserine (PS). Fluorescamine derivatization of external aminophospholipids revealed that PS, but not phosphatidylethanolamine, was externalized. The vitamin E analogue, 6-hydroxy-2,2,5,7,8-pentamethylchromane (PMC), inhibited overall oxidation of cis-PnA in phospholipids by more than 85%. Not all phospholipids, however, were equally protected. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and sphingomyelin were nearly completely protected by PMC, while oxidation of PS was unaffected in whole living cells. The insensitivity of PS to PMC was not an intrinsic property because PMC protected all lipids equally during AMVN oxidation of liposomes prepared from cis-PnA-labeled cells. The potential role for PS oxidation in apoptosis was further suggested by the faithful execution of apoptosis following coexposure of cells to AMVN and PMC.

Bcl-2 inhibits selective oxidation and externalization of phosphatidylserine during paraquat-induced apoptosis.

Oxidative stress is a potential component of the final common pathway leading to apoptosis following many diverse stimuli. Here, we document that the oxidant paraquat caused apoptosis in mouse 32D cells. We examined early paraquat-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid (cis-PA) into phospholipids and high-performance liquid chromatography separation of specific phospholipid classes. Paraquat induced peroxidation of cis-PA primarily in phosphatidylserine (PS) and to a lesser extent in phosphatidylinositol (PI) within 2 h. The selective oxidation of PS occurred before signs of cytotoxicity and preceded the externalization of PS as assessed by annexin V binding. Overexpression of Bcl-2 afforded significant protection against paraquat-induced apoptosis, early PS and PI oxidation, and PS externalization but not the ultimate formation of high-molecular-weight DNA fragments. Therefore, both selective phospholipid peroxidation and DNA damage occurred after paraquat exposure, but only the former was specifically associated with apoptosis. We suggest Bcl-2 may inhibit oxidant-induced apoptosis by preventing the peroxidation of specific membrane phospholipids.

Selective Modifications of the Phospholipid Fatty Acid Composition in Human Platelet Membranes Using Nonspecific and Specific Lipid Transfer Proteins

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

Effect of specific phospholipid molecular species incorporated in human platelet membranes on thromboxane A2/prostaglandin H2 receptors

The incorporation of albumin-bound docosahexaenoic acid (22:6n-3), but not linoleic acid (18:2n-6), into cellular phospholipids inhibits platelet aggregation induced by the thromboxane analogue U46619. [3H]U46619 specific binding to thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, as well as specific binding of the antagonist [3H]SQ29548 to these sites were also decreased in these modified cells (P. G., Swann et al. 1990. J. Biol. Chem. 265: 21692-21697). More than 80% of the 22:6n-3 incorporated in these cells was esterified in the various endogenous phospholipid classes and the remaining was found in neutral lipids and in the unesterified fatty acid pool. In this study, we determined whether the effects observed could be attributed to the esterification of 22:6n-3 in phospholipids and whether the 22:6n-3 biological activity might depend on its esterification in specific phospholipid classes. Therefore, pure phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species were transferred to platelet membranes, using lipid transfer proteins. PC and PE containing palmitate (16:0) and 22:6n-3 or 16:0 and 18:2n-6 at position sn-1 and sn-2, respectively, were incorporated into membranes only at the expense of the corresponding endogenous phospholipid class, by an apparent exchange process. When such modified membranes were tested for specific binding of U46619 and SQ29548, a significant decrease of the receptor site affinity was only observed in membranes highly enriched with 1-palmitoyl-2-docosahexaenoyl-glycerophosphocholine (16:0/22:6-GPC). Fluidity parameters measured by electron spin resonance of 5- and 16-nitroxy-stearic acids were not significantly different in membranes enriched with 16:0/22:6-GPC relative to those enriched with 16:0/18:2n-6-GPC, arguing against a generalized perturbation of the membrane due to 22:6n-3 incorporation. We conclude that molecular species of PC with 22:6n-3 at the sn-2 position can affect TXA2/PGH2 receptors. The selectivity of the inhibitory effect of PC containing 22:6n-3 is discussed.

Analysis of complex mixtures of phospholipid classes from cell membranes using two-dimensional thin-layer chromatography and scanning laser densitometry

Increasing recognition of the important roles served by membrane phospholipids in cellular metabolic and signal transduction processes has stimulated interest in examining potential phospholipid abnormalities in patients with psychiatric disorders. This report describes a method, based on several novel modifications of existing techniques, for concurrently analyzing nanomolar amounts of nine phospholipid classes in a single aliquot of membrane extract. With this method, diverse phospholipid classes are first separated by two-dimensional thin-layer chromatography, and then determined using two-dimensional scanning laser densitometry. The method is able to quantitate even small amounts of specific phospholipid classes, corresponding to < 10 ng of lipid phosphorus. The sensitivity of this method allows it to be readily applied to clinical studies involving membranes from cell types that are obtainable only in small quantities.

Direct determination of phospholipid structures in microorganisms by fast atom bombardment triple quadrupole mass spectrometry

When phospholipids ionized by fast atom bombardment undergo collisionally induced dissociation (CID), they cleave at specific bonds between the functional groups contained on the lipid. These cleavages are common to all classes of phospholipids. By taking advantage of this fact, a general scheme has been developed that uses a triple-quadrupole mass spectrometer to rapidly characterize the phospholipid content and structures present in crude lipid extracts. This scheme is based on fast atom bombardment ionization of a crude lipid extract and on the combination of positive-ion neutral-loss and parent scans and negative-ion daughter scans. Neutral-loss and parent scans provide independent diagnostic mass spectra for each of many specific phospholipid classes, while daughter scans provide the emperical formulas and positions of the fatty acyl constituents on each phospholipid. An automated tandem mass spectrometry (MS/MS) instrument can perform an extensive phospholipid screening on a single sample. A useful mass profile of the phosphatidylethanolamine species present in a 1-pg sample of mixed phospholipids (equivalent to ten Escherichia coli cells) has been obtained. The spectra are reproducible and proportional to concentration over at least the five-logarithm range of cell concentrations studied. A rapid extraction procedure combined with the automated instrument control program produces profiles of the phospholipid classes, along with fatty acyl empirical formulas and position information, on selected phospholipid species, in a few minutes, from a single sample.