Ether glycerophospholipids of gills of two pacific crabs,Cancer antennarius andPortunus xantusi

Phospholipids and sterols constituted 70 and 20%, respectively, of the total lipids of the gills of two crabs,Cancer antennarius andPortunus xantusi. Phosphatidylcholine (46–55% of the total phospholipid phosphorous) and phosphatidylethanolamine (24–25%) were the principal phospholipids present. In both species 1′-alkenyl glycerols were present in about 20% of the phospholipid molecules but were not detected in the neutral gill lipids. The total ether phospholipids ofC. antennarius gills contained 62% 1-(1′-alkenyl) groups, with the remainder probably being 1-alkyl moieties. Total gill plasmalogen contents were in the range of 163–184 μmol/g lipid, 82–87% of which was in the phosphatidylethanolamine fraction in both crab species.     

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.     

Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

Fatty acids in phospholipids isolated from human red cells

Total lipids of packed erythrocytes from healthy men 22 to 25 years old were extracted with chloroform-methanol mixture. Phospholipid classes were separated from neutral lipids and pigments on a silicic acid column. Phosphatidyl inositol (PI) was freed of its contaminants phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) on an aluminum oxide column. Additional silicic acid columns with modified solvent systems were needed for complete separation of other overlapped phospholipid classes. The identification of phospholipids in each eluted fraction was accomplished by TLC, using the appropriate spray tests and reference compounds, and confirmed on each of the isolated phospholipids by IR spectrophotometry. The total content of phospholipids as determined by phosphorus analysis was found to be 2.63 mg/ml of packed cells. These phospholipids were found to have the following composition (in per cent of total phospholipid): PI, 2.3; PE, 13.4; ethanolamine plasmalogen (EP), 14.5; PS, 3.9; lecithin (L), 34.2; choline plasmalogen (CP), 1.4; sphingomyelin (Sph), 28.4 and lysolecithin (LL), 1.7. The fatty acid composition of each phospholipid was determined by GLC. The average number of double bonds per fatty acid in the isolated phospholipids was found to be as follows: PI, 1.5; PE, 1.9; EP, 3.6; PS, 2.1; L, 1.0; CP, 2.0; Sph, 0.2 and LL, 0.5. The positional distribution of fatty acids in both L and PE was ascertained by selective enzymatic hydrolysis with phospholipase A. Saturated fatty acids of L were esterified predominantly in the α′-position, whereas in PE only 63.9 mole per cent of the saturated fatty acids were found in this position. Presented in part at the AOCS Meeting in Los Angeles, April 1966. Dept. of Health, Education and Welfare, USPHS.     

A complete separation of lipids by three-directional thin layer chromatography

A novel three-directional thin layer chromatography (TLC) method is reported by which all the polar and neutral lipids are isolated on a single TLC plate. Following resolution of the phospholipids by two-directional TLC, lipids are visualized by ultraviolet light after spraying with 2′,7′-dichloro-fluorescein. A line is drawn across the plate, parallel to the second direction of development, separating the resolved phospholipids and the neutral lipids concentrated along the solvent front. The TLC plate is then chromatographed in the reverse direction of the second development to resolve the neutral lipids. By exposing the lipids to HCl fumes after the first development, the plasmalogen content of the lipids may also be determined. This new technique is rapid and lends itself to qualitative and quantitative analyses of total lipids. Contribution no. 1163 from the Animal Research Centre.     

Meningioma phospholipid profiles measured by31P nuclear magnetic resonance spectroscopy

Fourteen cases of intracranial meningioma were characterized after chloroform/methanol extraction by³¹P nuclear magnetic resonance (NMR) spectroscopy at 202.4 MHz. Each phospholipid class detected in the extracts was identified and quantitated in terms of its molar percentage relative to the total phospholipids measured. The following phospholipids were assayed by³¹P NMR: phosphatidylglycerol, phosphatidic acid, diphosphatidylglycerol, ethanolamine plasmalogen, phosphatidylethanolamine (PE), lysophosphatidylinositol, phosphatidylserine, sphingomyelin, lysophosphatidylcholine (LPC), phosphatidylinositol (PI), sphingosylphosphorylcholine and phosphatidylcholine. In addition, two unidentified phospholipids were detected with resonances at 0.13 and −0.78 ppm, respectively. Three distinct types of spectra were obtained on the extracts and grouped accordingly for comparison purposes. Type 1 tumors showed unusual³¹P NMR profiles with low levels of PE and PI and elevated levels of LPC; type 2 tumors were characterized by low levels of the ethanolamine phospholipids and near equivalent levels of PI and LPC. The spectra of type 1 and type 2 tumors were characteristic of degenerative cells that lacked membrane permeability associated with loss of ethanolamine plasmalogen in the presence of significant phospholipid turnover. Meningiomas belonging to the third spectral type showed characteristics similar to those of normal tissues with normal levels of PE and ethanolamine plasmalogen, as well as very low levels of LPC relative to PI. Type 3 tumors lacked the characteristic signs of degeneration noted in type 1 and type 2 tumors. The data corroborate and augmentin vivo spectroscopic findings reported earlier and demonstrate the value of³¹P NMR spectroscopic phospholipid analysis on lipid extracts for the characterization of meningiomas.     

Identification of diacylglycero-4′-O-(N,N,N-trimethyl)homoserine in mushrooms

An unusual polar lipid component was found in some mushrooms and was isolated fromBoletus edulis. It has been identified as diacylglycero-4′-O-(N,N,N-trimethyl)-homoserine (DGTS) by its thin-layer chromatography (TLC) mobilities and staining behavior as well as by means of infrared (IR) and nuclear magnetic resonance (¹H NMR) spectroscopy and mass spectrometry. Identification of this lipid in mushrooms indicated that DGTS is widely distributed among a diverse group of lower plants and may have chemotaxonomic significance for fungi. It is interesting that DGTS is a component of some edible mushrooms commonly consumed in food.     

Regulation of phosphatidylinositol turnover,calcium metabolism and enzyme secretion by phorbol dibutyrate in neutrophils

The action of the tumor promoter, phorbol 12,13-dibutyrate (PDBu), on rabbit peritoneal and human neutrophils is associated with stimulation of¹⁴C-arachidonic acid incorporation into phospholipids within 1–2 min. Stimulated¹⁴C-arachidonate incorporation was relatively selective for phosphatidylinositol (PI) in rabbit neutrophils. In contrast, the secretory response of human neutrophils to PDBu coincided with stimulated label incorporation into phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA) and PI. Significant increases in label incorporation were observed with PDBu concentrations as low as 2 nM, and the dose response of stimulated label incorporation paralleled that of evoked lysozyme secretion. A parallel, but partial, inhibition of PDBu-stimulated PI labeling and enzyme release was observed after exposing rabbit neutrophils to calcium-deprived medium, whereas calcium deprivation failed to significantly depress either of these stimulant actions of PDBu in human neutrophils. Further, in rabbit neutrophils PDBu elicited an increase in cell associated⁴⁵Ca. However, PDBu was unable to promote the incorporation of³²P orthophosphate into PI or enhance phospholipase A₂ activity in broken cells. These findings suggest that one expression of the interaction between phorbol esters and their receptors on neutrophils involves the turnover of arachidonic acid in phospholipids. This stimulated turnover of arachidonate may be a critical step in the cascade of events associated with neutrophil activation.     

Effect of FA chain length on normal- and reversed-phase HPLC of phospholipids

Forty-seven saturated synthetic diacyl PA, PC, PE, PG, and PS and five unsaturated diacyl phospholipids (PL) underwent normal- and reversed-phase (RP) HPLC with isocratic isopropanol/hexane/water (5∶4∶1) and methanol/chloroform/acetonitrile/water (79.5∶9∶8∶3.5) mobile phases, respectively. For normal-phase HPLC, capacity factors (k′ _i ) decrease with chain length (n) of the two identical PL FA residues, whereas the opposite occurs with RP (C₁₈)-HPLC. Plots of In k′ _i vs. n for individual PL classes are in general curved, violating the linear free-energy relationship. For PL of the same n but with different head groups, k′ _i with normal-phase HPLC varies as PE<PG<PA<PS<PC, except when n≥16, when the order is PE<PS≈PA≈PG<PC. For RP-HPLC, the order of k′ _i values is PG<_A≈PS≤PC≈PE until n≥16, when it is PA≈PG<PS≪PC≈PE. With normal-phase HPLC, k′ _i values of PL with unsaturated FA of n=18 are ordered as PE<PA<PC. Increasing degrees of unsaturation lead to increasing k′ _i .     

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium ofictalurus punctatus in vitro

The catfish,Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [³²P]phosphate was incubated with minced pieces of epithelium from the taste organ ofI. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC),phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20–120 min time period. Upon addition of 1,2-di-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine to the medium,¹⁴C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However,¹⁴C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1′-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the¹⁴C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids. The PC fraction was maximally labeled by 40 min. Using the appropriate radiolabeled substrates, lysophosphatidylcholine phospholipase A₁ and phosphatidylcholine phospholipase D activities were detected in this tissue. Very low activity of a phosphatidylcholine phospholipase A₂ was observed. The experiments indicate that there are active and rapid exchange, degradation, synthesis and scavenger pathways of phospholipids in the taste organ of this animal, and suggest that phospholipases A₁ and D-type activities are primarily responsible for the rapid breakdown of LPC and PC.     

Phospholipids inDrosophila heads: Effects of visual mutants and phototransduction manipulations

A procedure was developed to label phospholipids inDrosophila heads by feeding radioactive phosphate (³²P_i). High-performance thin-layer chromatography showed label incorporation into various phospholipids. After 24 h of feeding, major phospholipids labeled were phosphatidylethanolamine (PE), 47%; phosphatidylcholine (PC), 24%; and phosphatidylinositol (PI), 12%.Drosophila heads have virtually no sphingomyelin as compared with mammalian tissues. Notable label was in ethanolamine plasmalogen, lysophosphatidylethanolamine, lysophosphatidylcholine and lysophosphatidylinositol. Less than 1% of the total label was in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Other lipids labeled included phosphatidylserine, phosphatidic acid and some unidentified lipids. A time course (3–36 h) study revealed a gradual decrease in proportion of labeled PI, an increase in proportion of labeled PC and no obvious change in labeled PE. There were no significant differences in phospholipid labeling comparing theno receptorpotential (norpA) visual mutant and wild type under lightvs. dark conditions. However, overall³²P labeling was higher in the wild type fed in the light as compared to the dark and tonorpA either in light or dark. This suggests that functional vision facilitates incorporation of label. Differences in phospholipid labeling were observed between young and aged flies, particularly in lysophospholipids and poly-PI, implicating phospholipase A₂ function in recycling. Manipulations such as theouterrhabdomeresabsent andeyesabsent mutants and carotenoid deprivation failed to yield notable differences in phospholipid labeling pattern, suggesting that phospholipids important to vision may constitute only a minor portion of the total labeled pool in the head.     

PGE<Subscript>2</Subscript> Release from Tryptase-Stimulated Rabbit Ventricular Myocytes is Mediated by Calcium-Independent Phospholipase A<Subscript>2</Subscript>γ

Inflammation is associated with cardiovascular disease, including myocardial infarction, atherosclerosis, myocarditis and congestive heart failure. Mast cells have been implicated in inflammation, but their precise role in cardiac inflammation remains unclear. Mast cells contain a variety of pre-formed granule-associated mediators, including tryptase. We have previously demonstrated that the majority of the phospholipase A₂ (PLA₂) activity in isolated rabbit ventricular myocytes is membrane-associated, calcium-independent and selective for plasmalogen phospholipids. We hypothesized that tryptase stimulation of rabbit ventricular myocytes would increase iPLA₂ activity, leading to increased arachidonic acid and prostaglandin E₂ (PGE₂) release. Isolated rabbit ventricular myocytes were stimulated with tryptase and iPLA₂ activity, arachidonic acid and PGE₂ release were measured. Tryptase stimulation increased iPLA₂ activity after 5 min. Activation of iPLA₂ was accompanied by increased arachidonic acid and PGE₂ release in tryptase-stimulated myocytes. However no increase in platelet activating factor was observed with tryptase stimulation. To distinguish between different iPLA₂ isoforms in the myocardium, we pretreated ventricular myocytes with the (R)- and (S)-enantiomers of bromoenol lactone (BEL) to selectively inhibit iPLA₂γ and β respectively. Pretreatment with (R)-BEL resulted in complete inhibition of tryptase-stimulated iPLA₂ activity, arachidonic acid and PGE₂ release, suggesting the iPLA₂γ is the predominant myocardial isoform activated by tryptase. These studies demonstrate that PGE₂ release from tryptase stimulated rabbit ventricular myocytes is mediated primarily by iPLA₂γ.     

Identification of carrot inositol phospholipids by fats atom bombardment mass spectrometry

Inositol phospholipids from carrot cell membranes grown in suspension cultured were purified by thinlayer chromatography (TLC) or column chromatography and tentatively identified by co-migration on TLC with animal inositol phospholipid standards. For more rigorous chemical characterization, carrot inositol phospholipids were then analyzed by negative ion fast atom bombardment mass spectrometry (FABMS). One phosphatidylinositol (PI), two lysophosphatidylinositols (LPI), and one phosphatidylinositol monophosphate (PIP) were identified in the carrot samples by the observation of ions [M-H]⁻ and numerous fragment ions in the negative FAB mass spectra. MS/MS analysis were carried out to obtain further structural information of these phospholipids using a double-focusing mass spectrometer in which the magnetic sector (B) and the electrostatic analyzer (E) were scanned at a constant ratio (B/E). These B/E linked scans provided fragment ions of selected precursor ions while eliminating matrix and other contaminating ions. No molecular ions were detected for lysophosphatidylinositol monophosphate (LPIP) or phosphatidylinositol bisphosphate (PIP₂), but fragment ions corresponding to these structures were observed. The primary fatty acids present in the carrot inositol phospholipids were linoleic (18∶2) and palmitic (16∶0) acids, whereas animal lipids contained arachidonic (20∶4), stearic (18∶0), linoleic, and palmitic acids. The only phosphatidylinositol found in carrot cells was palmitoyl linoleoyl PI.     

Lipid composition and erucic acid in rat liver cells in culture

Erucic acid (Δ¹³-docosenoic acid) was added to fetal calf serum, then fed to rat liver epithelial cells in culture, and uptake measured at intervals over 24 hr. During the first 6 hr. of incubation, uptake of the docosenoic acid was 21 nmoles/hr/mg protein in 7-day cells, and 15 mmoles/hr/mg protein in 14-day cells. Of¹⁴C-labeled erucic acid taken up by the cells in 24 hr, radioactivity measurements showed 60% of the total lipid¹⁴C activity derived from [1-¹⁴C] 22∶1 in neutral lipid (NL) and 40% in phospholipid (PL); whereas 55% of lipid¹⁴C activity was in NL and 45% in PL when the substrate was [14-¹⁴C] 22∶1. Within the NL fraction, 75% of¹⁴C activity derived from [1-¹⁴C] 22∶1 was in triglyceride (TG) and 11% in cholesterol (CHL), while 79% was in TG and 6.5% in CHL when the substrate was [14-¹⁴C] 22∶1. Triglycerides and cholesteryl esters accumulated in the cells during incubation with erucic acid. Among phospholipids separated by thin layer chromatography, 75% of¹⁴C activity was in lecithin (PC), 10% in phosphatidylethanolamine (PE), 5% in sphingomyelin (SPH), and 1% or less in cardiolipin (DPG). The highest specific activity (SA) was in PC, followed by SPH and PE. Incubation with erucic acid altered fatty acid composition of PC, PE, and SPH, although amounts of phospholipids were unaffected. Gas liquid chromatography analyses detected 18% erucic acid in PC, 2% in PE, and 4–5% in SPH.     

Glyceryl ethers in insects: Biosynthesis of ethanolamine phosphoglycerides containing alkyl and alk-1-enyl glyceryl ether linkages

When¹⁴C-labeled acetate, fatty acids or fatty alcohols were injected into or fed to the tobacco budworm, acyl, alkyl and alk-1-enyl moieties of the phospholipids incorporated radioactivity. Fatty acids were the principal precursor in acyl bond formation and fatty alcohols in the synthesis of alkyl and alk-1-enyl glyceryl ethers. Detailed analysis of the etherlinked phosphoglycerides revealed that most of the radioactivity was in the ethanolamine phosphoglycerides, and very little¹⁴C was found in the choline phosphoglycerides. In experiments of a short duration, the alkyl glyceryl ethers incorporated more radioactivity than the alk-1-enyl glyceryl ethers. The reverse was found with long term experiments, when the alk-1-enyl ethers had higher radioactivity. In addition to demonstrating the synthesis of ether-linked ethanolamine phosphoglycerides, the data suggested that fatty alcohols and acids were interconverted by insects and that the alk-1-enyl ethers were derived from the alkyl ethers. Presented at the AOCS Meeting, Houston, May 1971. The following abbreviations and terminology will be used: PE, PC, PI and PS for the generic terms ethanolamine, choline, inositol and serine phosphoglycerides, respectfully. Alkyl glyceryl ether for 1-alkyl-2-acyl-sn-glycerol-3-phosphoryl-, and alk-1-enyl glyceryl ether for 1-alk-1′-enyl-2-acyl-sn-glycerol-3-phosphoryl-(commonly called plasmalogen). These are adapted from the tentative rules published inJ. Lipid Res. 8:522–528 (1967).     

TLC and <Superscript>31</Superscript>P-NMR Analysis of Low Polarity Phospholipids

High-performance TLC and ³¹P-NMR were assessed as methods of observing the presence of numerous low polarity phospholipids: bis-phosphatidic acid (BPA), semi-lyso bis-phosphatidic acid (SLBPA), N-acyl phosphatidylethanolamine (NAPE), N-(1,1-dimethyl-3-oxo-butyl)-phosphatidylethanolamine (diacetone adduct of PE, DOBPE), N-acetyl PE, phosphatidylmethanol (PM), phosphatidylethanol (PEt), phosphatidyl-n-propanol (PP), phosphatidyl-n-butanol (PB). Both techniques are non-discriminative and do not require the prior isolation of individual lipids. It appears that 2D TLC is superior to ³¹P NMR in the analysis of low polarity phospholipids. All phosphatidylalcohols were well separated by 2D TLC. However, some compounds which can present difficulty in separation by 2D-TLC (e.g., SLBPA and NAPE; or DOBPE and N-acetyl PE) were easily distinguished using ³¹P NMR so the methods are complimentary. A disadvantage of 2D TLC is that Rf values can vary with different brands and batches of TLC plates. The chemical shifts of ³¹P NMR were less variable, and so a library of standards may not be necessary for peak identification. Another advantage of ³¹P NMR is the ease of quantification of phospholipids. The applicability of the methods was tested on natural extracts of fish brain and cabbage stem.     

Phospholipid studies of marine organisms: New branched fatty acids fromStrongylophora durissima

The phospholipids of the spongeStrongylophora durissima were analyzed. The major phospholipids present were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI). The major fatty acid components of the phospholipids consisted of short chain (C₁₄−C₁₉) and very long chain (C₂₅−C₃₀) “Demospongic” acids. Three novel branched Δ⁵ monounsaturated acids,Z-19-methyl-5-pentacosenoic,Z-19-methyl-5-hexacosenoic andZ-19-methyl-5-heptacosenoic acids were encountered in the sponge. The 3-saturated counterparts of these compounds, 19-methylpentacosanoic, 19-methylhexacosanoic and 19-methylheptacosanoic acids, as well as 19-methylpentacosanoic and 20-methyloctacosanoic acids also are hitherto undescribed acids present in the sponge. Trace amounts of 2 very long chain acids also were detected and their structures tentatively assigned as 19,21-dimethylheptacosanoic and 20,22-dimethyloctacosanoic acids. The distribution of these fatty acids according to phospholipid head groups also was described.     

Alterations in heart and kidney membrane phospholipids in hypertension as observed by 31P nuclear magnetic resonance

Abnormalities of phospholipids in hypertension have previously been described in human erythrocyte, platelet, and plasma lipoproteins. Since the heart and kidney are adversely affected by hypertension, we investigated possible alterations in their membrane phospholipids, which could play a role in the derangement of intracellular ion balance widely observed in hypertension. The phospholipid compositions of heart and kidney from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were determined by using ³¹P nuclear magnetic resonance (NMR) spectroscopy. Absolute contents of all phospholipids in hypertensive hearts and kidneys were significantly higher than in normotensive hearts and kidneys. Expressed as a fraction of total phospholipid, cardiolipin (CL) and phosphatidylethanolamine plasmalogen (PEp) were significantly increased in SHR hearts compared to WKY hearts (CL and PEp were 7.95±0.22% and 13.16±0.35% in SHR vs. 7.01±0.20% and 11.19±0.42% in WKY rats, P<-0.05), but phosphatidylethanolamine (PE) and phosphatidylcholine (PC) were significantly decreased in SHR (PE and PC were 22.46±0.37% and 44.81±0.43% in SHR vs. 24.02±0.44% and 46.01±0.50% in WKY rats, p≤0.05). In the phospholipids extracted from rat kidneys, the percentage of PE was significantly higher for SHR than for WKY rats (20.37±0.60% vs. 18.43±0.37%, P≤0.05), while PEp and phosphatidylserine (PS) were significantly lower for SHR (PEp and PS were 10.22±0.36% and 8.42 ±0.28% in SHRs vs. 11.29±0.36% and 9.71±0.40% in WKY rats, P≤0.05). The above alterations in phospholipid composition might contribute to the higher oxygen consumption in the hypertensive heart and abnormal intracellular ion concentrations and ion transport in the heart and the kidney in hypertension.     

31P NMR of tissue phospholipids: Competition for Mg2+, Ca2+, Na+ and K+ cations

Phosphatidylcholine (PC), phosphatidylethanolamine (PE), ethanolamine plasmalogen (EPLAS), sphingomyelin (SPH), phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidic acid (PA) were dispersed together in Cs(ethylenedinitrilo)tetraacetic acid-scrubbed chloroform/methanol solution, and high resolution³¹P nuclear magnetic resonance spectra were recorded. In separate titration experiments, Mg²⁺ and Ca²⁺ were added to the dispersed phospholipid mixture to determine the relative interaction potentials of each of the phospholipids for each of the added cations. The association of cations with individual phospholipids was indicated by³¹P chemical-shift changes, signal broadening, signal quenching or a combination of these. The titrations revealed that CL had the highest, and PA the next highest, interaction potential for Mg²⁺ cations. In contrast, PS and PA had the highest, and CL the next highest, interaction potential for Ca²⁺. Considering only interactions with Ca²⁺ ions, the phospholipids can be divided into three distinct groups: PS and PA (high interaction potential); CL, PI and PG (intermediate interaction potential); and EPLAS, PE, SPH and PC (essentially no interaction potential). The two phospholipids with the least interaction potential for either of the alkaline-earth cations were PC and SPH. Na⁺ and K⁺ ion interactions with PA, CL, PI and PG were unique and resulted in positive chemical-shift changes relative to the chemical shifts in the presence of Cs⁺ ions. Relative to both Cs⁺ and K⁺ ions, chemical shifts in the presence of Na⁺ ions were deshielded δ>0.1 ppm in the order PA>CL>PI>PG.     

Polyphosphoinositides and the shape of mammalian erythrocytes

The relationship between polyphosphoinositide and phosphatidic acid (PA) metabolism and Mg-ATP dependent shape and viscosity changes in erythrocyte ghosts from four mammalian species was examined. Ghosts prepared from rabbit, dog, human and guinea pig erythrocytes were transformed from echinocytes to discocytes within 15 min in the presence of 1 mM Mg-ATP at 25 C. In all species these Mg-ATP shape transformations were associated with a 30–45% decrease in the specific viscosity of the ghost suspensions. Mg-ATP induced a second transformation of discocytic ghosts to cup shape forms without a further decrease in viscosity. A considerable species variation in the rates of Mg-ATP dependent viscosity and shape changes and incorporation of³²P into phosphatidylinositol-4′ phosphate (PIP), phosphatidylinositol-4′5′bisphosphate (PIP₂) and especially PA form MG-[γ ³²P]-ATP in ghosts was found. However, the rates of Mg-ATP dependent synthesis of PIP and PIP₂ and shape and viscosity changes in each species were of the same magnitude. Ca²⁺ or neomycin strongly inhibited PIP labeling and Mg-ATP shape and viscosity changes in ghosts of the different species. Ca²⁺ or neomycin usually increased or had little effect on³²P incorporation into PA and PIP₂. The possibility that Mg-ATP-induced changes in erythrocyte membrane shape and deformability are dependent on increases in membrane PIP and PIP₂ is discussed.