Densitometric quantitation of individual phospholipids from natural sources separated by one-dimensional thin-layer chromatography

A method for the quantitation of small amounts of phospholipids derived from biological sources is described. Total phospholipid is determined by mineralization followed by the estimation of liberated phosphate by means of malachite green. The main phospholipid species are separated by one-dimensional thin-layer chromatography. The individual phospholipids are detected by charring with CuSO4/H3PO4. They may be directly quantitated by scanning the thin-layer chromatography plates with a laser densitometer.     

Separation and quantitation of phospholipids and lysophospholipids by high-performance liquid chromatography

We describe a comprehensive approach to the separation, quantitation, and characterization of phospholipids and lysophospholipids present in complex biological samples. The central feature is a normal-phase HPLC separation of individual phospholipid and lysophospholipid classes. In this single chromatographic step, phospholipids and lysophospholipids are separated and recovered for quantitation by organic phosphate assay and characterization by acyl-group composition. Recovery of phospholipids and lysophospholipids from HPLC averages 80-90%. Isolated phospholipid and lysophospholipid fractions are available for separation of individual molecular species by second-dimension reverse-phase HPLC and characterization of individual molecular species by mass spectrometry.     

32P incorporation into different membranous structures separated from rat cerebral cortex

Abstract— The time course of incorporation, between 3 hr and 16 days, of ortho[³²P]phosphate into different membranous structures isolated from the rat cerebral cortex was studied. After subarachnoideal administration into the CSF it was found that myelin, mitochondria, microsomes and purified nerve-ending membranes and synaptic vesicles incorporate ³²P at the same rate. Most of the individual phospholipids of the synaptic vesicles and nerve-ending membranes also have similar rates of incorporation. Only phosphoinositides and/or phosphatidylserine may have a more rapid metabolism. The incorporation of ³²P into phosphoproteins follows a different pattern from that of the phospholipids. The intraperitoneal route is less effective in the ³²P incorporation and differences among the fractions may be found. These results are discussed in relation to the problem of the blood-brain barrier to phosphate and to the labelling of individual phospholipids in the different membranes.     

Restoration by phospholipids of dolichol pyrophosphate N-acetylglucosamine synthesis in delipidated rat lung microsomes

The effects of phospholipids on the reaction catalyzed by UDP-GlcNAc:dolichol phosphate GlcNAc-1-phosphate transferase have been studied with delipidated rat lung microsomes. Deoxycholate-solubilized enzyme was depleted of measurable phospholipid by either gel filtration on Sephadex G-100 or affinity chromatography on pentyl-agarose. The latter procedure also removed nucleotide and sugar nucleotide hydrolases. Delipidated protein fractions were devoid of GlcNAc-1-phosphate transferase activity unless supplemented with phospholipids. Maximal recovery of enzyme activity was obtained with an approximate 1:1 weight ratio of phosphatidylglycerol:phosphatidylcholine, with the observed rate being synergistic as compared to rates observed for each individual phospholipid. Variable recoveries of enzyme activity were obtained with mixtures containing other acidic phospholipids and phosphatidylcholine. Enzyme activity in the fraction eluted from pentyl-agarose could be recovered, after removal of Triton X-100, with sedimented phospholipid vesicles. Significant stabilization of enzyme activity associated with the phospholipid vesicles was obtained by the inclusion of dolichol phosphate.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline.

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.     

Phospholipid metabolism during changes in the proportions of membrane-bound respiratory pigments in Haemophilus parainfluenzae

After a transition from high to low oxygen tension, there was a twofold to 50-fold increase in the content of membrane-bound respiratory pigments of Haemophilus parainfluenzae, and there were concurrent changes in the metabolism of the membrane phospholipids: (i) a twofold decrease in the rate of turnover of the phosphate in all the phospholipids; (ii) a shift from simple one-phase, linear incorporation of phosphate into phospholipids to a complex biphasic incorporation of phosphate into phospholipids; and (iii) an increase in the total phospholipids with a slight increase in the proportion of phosphatidylglycerol (PG) and a slight decrease in the proportion of phosphatidylethanolamine (PE). Changes in the rates of incorporation of phosphate into the phospholipids occurred without a change in the rate of bacterial growth. When the compensatory adjustment of the proportions of the respiratory pigments reached a steady state, the total phospholipid, the rate of incorporation of phosphate into phospholipids, and the proportion of PG fell. At steady-state proportions of cytochromes, the proportion of PE and the rate of turnover of the phosphate in the phospholipids increased. All through an incorporation experiment of 1.5 divisions, the specific activity of the phosphate of PG was twice that of phosphatidic acid (PA). The phosphate of PG turned over 1.2 to 1.5 times more rapidly than the phosphate of PA in cells with high and low cytochrome levels. If the PA was an accurate measure of the precursor for the cytidine-5′-diphosphate-diglyceride, which in turn was the precursor of all the lipids, then the results of these experiments suggested that exchange reactions, in addition to synthesis from PA, were involved in phospholipid metabolism. These reactions were more sensitive to changes in oxygen concentration than was the growth rate.     

Compositional and molecular species analysis of phospholipids by high performance liquid chromatography coupled with chemical ionization mass spectrometry

High performance liquid chromatography (HPLC) was combined with chemical ionization mass spectrometry (CIMS) by the use of a moving-belt interface. The technique was employed for the analysis of naturally occurring phospholipids. Positive and negative ion mass spectra of various phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin were obtained in the chemical ionization mode with ammonia or methane as the reagent gas. Specific ions for individual phospholipid "bases" were identified. These ions were used in specific ion monitoring of the phospholipids during HPLC-CIMS. CIMS of each phospholipid also provided extensive information on the molecular species of the individual class of phospholipids. Relative abundance of different molecular species of each phospholipid as determined by CIMS agreed well with the results obtained by gas-liquid chromatography. Rat brain phospholipids were analyzed by HPLC-CIMS in about 15 minutes. Routinely, about 5 micrograms of individual phospholipid was analyzed by HPLC-CIMS, however, with specific ion monitoring the method provides a detection capability at the subnanogram level.     

Amphiphilic cationic drugs and phospholipids influence the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fraction of untreated rats

The influence of amphiphilic drugs and phospholipids on the activities of beta-galactosidase and beta-glucosidase from liver lysosomal fractions of untreated rats, isolated by affinity chromatography using castor bean lectins, was studied in vitro. Chloroquine (93 microM) inhibited beta-galactosidase activity by about 30%, while O,O'-bis(diethylaminoethyl)hexestrol showed no inhibitory effect. Neutral phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelin) inhibited the enzyme slightly, while the enzyme activity was drastically reduced in the presence of acidic phospholipids (phosphatidylinositol, phosphatidylserine, bis-(monoacylglycero)phosphate). Lysosomal beta-glucosidase was strongly inhibited by chloroquine and O,O'-bis(diethylaminoethyl)-hexestrol. The neutral phospholipids showed only a moderate inhibitory effect, whereas the acidic phospholipids were stimulators. Bis(monoacylglycero)phosphate was by far the best stimulating compound.     

The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

The turnover of myelin phospholipids in the adult and developing rat brain

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.     

Interaction of europium(III) with phospholipid vesicles as monitored by laser-excited europium(III) luminescence

The technique of laser-excited Eu(III) luminescence was applied to monitor Eu(III) binding to a variety of phospholipids. Eu(III) excitation spectra were similar with and without the presence of neutral phospholipids, while acidic phospholipids changed the spectrum in a concentration-dependent manner. Eu(III) appears to bind to the phosphate moiety with at least a 2:1 phospholipid:metal ion stoichiometry. Analysis of luminescence lifetimes reveals that only one or two waters of hydration are removed from Eu(III) by addition of neutral phospholipids, whereas acidic phospholipids and inorganic phosphate strip off all but one or two waters. Implications with regard to fusion and use of lanthanides as probes in membrane preparations are discussed.     

A simplified procedure for organic phosphorus determination from phospholipids

We describe an improved method for the determination of organic phosphorus from phospholipids. It is the combination of a very fast mineralization step followed by the estimation of liberated phosphate by means of malachite green. This method is accurate, simple, and sensitive.     

The effect of phosphate deficiency on membrane phospholipid composition of bean (Phaseolus vulgaris L.) roots

Plasma membranes were isolated from roots of bean (Phaseolus vulgaris L.) plants cultured on phosphate sufficient or phosphate deficient medium. The phospholipid composition of plasma membranes was analyzed and compared with that of the microsomal fraction. Phosphate deficiency had no influence on lipid/protein ratio in microsomal as well as plasma membrane fraction. In phosphate deficient roots phospholipid content was lower in the plasma membrane, but did not change in the microsomal fraction. Phosphatidylcholine and phosphatidylethanolamine were two major phospholipids in plasmalemma and microsomal membranes (80 % of the total). After two weeks of phosphate starvation a considerable decrease (about 50 %) in phosphatidylcholine and phosphatidylethanolamine in microsomal membranes was observed. The decline in two major phospholipids was accompanied by an increase in phosphatidic acid and lysophosphatidylcholine content. The effect of alterations in plasma membrane phospholipids on membrane function e.g. nitrate uptake is discussed.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

Phosphorylation of troponin I by protein kinase C: mechanism of inhibition by calmodulin and troponin C

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.     

Polymer-coated liposomes resistant to enzymatic hydrolysis

A polymerizable electrolyte, 2-aminoethyl 1,6-heptadien-4-yl phosphate (AEHDP), which has the same hydrophilic head group as naturally occurring phospholipids, was prepared. Five equivalents of AEHDP were added to a suspension of liposomes (closed bilayer vesicles made of phospholipids) and layered on the liposomes. After polymerization by UV irradiation, the resulting polymer-coated liposomes were resistant to hydrolysis of their constituent phospholipids by phospholipase A2.     

Hydrogen bonding between anhydrous cholesterol and phosphatidylcholines: an infrared spectroscopic study

Fourier transform infrared spectroscopy performed with a high pressure diamond anvil cell was used to study hydrogen bonding between anhydrous phosphatidylcholines and cholesterol at the molar ratio 4:1. The hydroxyl group of cholesterol which acts as a proton donor, engages in strong hydrogen bonding to the sn-2 chain carbonyl group of DMPC, DPPC and HPPC and in weak hydrogen bonding to the phosphate group of all these phospholipids. No evidence of hydrogen bonding between cholesterol and the sn-1 chain carbonyl group of DMPC and DPPC was found. From a comparison of the relative hydrogen-bond strengths between cholesterol or water and the sn-2 chain carbonyl and phosphate groups of all these phospholipids, it is predicted that in aqueous dispersions of cholesterol containing phospholipids, the hydrogen bond of cholesterol to the phosphate group would be replaced by that of water, while the hydrogen bond of cholesterol to the sn-2 chain carbonyl group would remain intact.