Galactolipid deficiency in the early pathogenesis of neuronal ceroid lipofuscinosis model Cln8mnd : implications to delayed myelination and oligodendrocyte maturation.

AIMS: CLN8 deficiency underlies one of a group of devastating childhood neurodegenerative disorders, the neuronal ceroid lipofuscinoses. The function of the CLN8 protein is currently unknown, but a role in lipid metabolism has been proposed. In human CLN8 diseased brains, alterations in lipid composition have been detected. To further investigate the connection of CLN8 to lipid metabolism, we characterized the lipid composition of early symptomatic Cln8-deficient mouse (Cln8(mnd)) brains. METHODS: For lipid profiling, Cln8(mnd) cerebral cortical tissue was analysed by liquid chromatography/mass spectrometry. Galactolipid synthesis was measured through enzyme activity and real-time mRNA expression analyses. Based on the findings, myelination and white matter integrity were studied by immunohistochemistry, stereological methods, electron microscopy and magnetic resonance imaging. The development of myelin-forming oligodendrocytes was also studied in vitro. RESULTS: Sphingolipid profiling showed a selective reduction in myelin-enriched galactolipids. The mRNA expression and activity of UDP-galactose:ceramide galactosyltransferase (CGT), the key enzyme in the galactolipid synthesis, was reduced in the Cln8(mnd) brain. Expression of oligodendrocyte markers suggests a maturation defect. The amount of myelin was reduced in 1-month-old Cln8(mnd) mice, but reached normal levels by 5 months of age. The level of Cln8 gene expression followed the developmental pattern of myelin formation and was high in primary oligodendrocytes. CONCLUSIONS: Taken together, these observations suggest that galactolipid deficiency and delayed myelin maturation characterize the early CLN8 disease pathogenesis through a maturation defect of oligodendrocytes.

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Subcellular localization of three degeneration-associated phospholipids in cultured hamster fibroblasts (BHK21 cells).

Subcellular localization of bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine was studied in normal and degenerating fibroblasts (BHK21 cells) by differential centrifugation. In the normal cells these lipids were highly enriched in the floating fraction consisting mainly of neutral lipid-rich lysosomes. They were also enriched in the mitochondrial fraction. In degenerating cells the high enrichment in the floating fraction was retained, but the other peak was displaced to the crude nuclear fraction. Subfractionation of the crude nuclear fraction indicated that these lipids were not enriched in the purified nuclei. Instead, their concentrations were relatively high in the other subfraction evidently enriched in the large secondary lysosomes characteristic for the degenerating cells. Neither in normal nor degenerating cells were these lipids enriched in the light mitochondrial fraction, where most of the smaller, and probably younger, lysosomes were found. On the basis of these results it is suggested that bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine are lysosomal in origin. It appears possible that they are specifically associated with the organelles representing the later stages in the lysosomal lifespan.

Conversion of phosphatidylglycerol lipids to bis(monoacylglycero)phosphate in vivo.

Liposomes containing either 32P-labeled diphosphatidylglycerol (cardiolipin) or 32P-labeled phosphatidyl-rac-(1)-glycerol were injected into the circulation of rats. Analysis of the liver lipids 2-3 after injection showed incorporation of the 32P label from both lipids to a lipid which had chromatographic properties identical with bis(monoacylglycero)phosphate. Stereochemical analysis of this lipid indicated that its backbone was sn-glycero-1-phospho-1'-glycerol. Cultured hamster fibroblasts (BHK cells) were incubated in a medium containing lyso[32P]phosphatidyl-rac-(1)-glycerol and the formation of radioactive lipids in the cells was followed. Bis(monoacylglycero) phosphate was the major 32P-labelled lipid formed: as much as 36.4% of the lyso[32P]phosphatidyl-rac-(1)-glycerol absorbed to the cells was converted to bis(monoacylglycero)phosphate. Similar results were obtained with lyso[32P]phosphatidyl-sn-1-glycerol as a precursor. Stereoanalysis of the bis(monoacylglycero)-[32P]-phosphate formed from either precursor indicated that this lipid was a derivative of sn-glycero-1-phospho-1'-glycerol. These results establish phosphatidylglycerol, diphosphatidylglycerol and lysophosphatidylglycerol as precursors of bis-(monoacyl-sn-glycero-1)phosphate in vivo. The mechanism of the conversion of lysophosphatidylglycerol to bis-(monoacyl-sn-glycero-1)phosphate was studied by using 32P,3H-labeled lysophosphatidyl-rac-(1)-glycerol as a precursor. Both labels were incorporated to bis(monoacylglycero)phosphate with similar efficiency, which suggests that rearrangement, rather than replacement, of the (originally acylated) sn-glycero-3-phospho moiety of the precursor is the essential reaction in the biosynthesis of the sn-glycero-1-phospho-1'-glycerol backbone of bis(monoacylglycero) phosphate.

Degradation of low density lipoprotein cholesterol esters by lysosomal lipase in vitro. Effect of core physical state and basis of species selectivity.

The effect of the physical state of low density lipoprotein (LDL) core and the selectivity of the degradation of LDL cholesterol esters (CEs) by the lysosomal acid lipase (LAL) in vitro were investigated. The physical state of LDL was modulated by varying temperature or the triglyceride content of the core. Normal LDL showed an abrupt increase of CE hydrolysis at 24 degrees C and another deviation occurred close to 36 degrees C. 1H-NMR measurements showed that these temperatures coincide with the onset and end temperatures of the LDL core lipid transition, respectively. Enrichment of LDL with triglycerides abolished the abrupt changes both in the CE hydrolysis and in the physical state of LDL lipids. These findings show that there is a correlation between the physical state of LDL lipids and the rate of LAL-mediated hydrolysis of the CEs in the particle. The relative rates of hydrolysis of different CE species were also compared. With native LDL, increasing the length of a saturated acyl chain from 14 to 20 carbons reduced the rate of degradation of CE modestly, while increasing acyl chain unsaturation increased the rate of degradation markedly. However, cholesterol oleate was hydrolyzed more slowly than cholesterol stearate. Essentially the same order of hydrolytic susceptibility was observed when the CE species were incorporated into triglyceride-enriched LDL, reconstituted high density lipoprotein particles or in detergent/phospholipid micelles. These results indicate that the selective hydrolysis of CE species in LDL is determined mainly by the ease with which the CE molecule can emerge from the surface layer reach the active site of LAL. Slower degradation of the more saturated CEs by LAL could lead, under certain conditions, to their accumulation in lysosomes and eventually, to cell death, lysis and deposition of crystalline, poorly mobilizable lipids to the arterial intima.

Glycerophospho-(N-acyl)-ethanolamine lipids in degenerating BHK cells.

The phospholipids, which accompany semilysobisphosphatidic acid from degenerating BHK cells, were identified as a mixture of glycerophospho-(N-acyl)-ethanolamine lipids. The identification was based on infrared spectroscopy, thin-layer chromatography and ethanolamine content of the intact lipids or their partial degradation products. Sequential treatments with mild acid and alkali revealed the presence of three different derivatives: the most abundant of these was the O-(1-alkenyl) ether derivative (plasmenyl-(N-acyl)-ethanolamine), which represented 55-60% of the total glycerophospho-(N-acyl)-ethanolamine lipids; the O-alkyl derivative (plasmanyl-(N-acyl)-ethanolamine) and the di-O-acyl derivative (phosphatidyl-(N-acyl)-ethanolamine) each represented about 20% of the total.

Parinaroyl and pyrenyl phospholipids as probes for the lipid surface layer of human low density lipoproteins.

A simple protocol employing lipid transfer proteins was developed to label human low density lipoprotein (LDL) in a controlled manner with parinaroyl and pyrenyl phosphatidylcholines. In order to study the lipid fluidity in the surface lipid layer of LDL, the temperature-dependence of both polarization (parinaroyl probes) and excimer to monomer (E/M) intensity ratio (pyrenyl probes) were analyzed. A series of pyrenyl phosphatidylcholines containing a pyrenyl fatty acid varying from 6 to 14 carbons in length at the sn-2 position were inserted into LDL to investigate the lateral distribution of different phosphatidylcholines in the lipoprotein surface at 37 degrees C. Both polarization and E/M vs. temperature plots displayed discontinuities in the region of 22-32 degrees C, which coincides with the melting of the neutral lipid core, indicating that the latter induces an ordered to more disordered phase transition in the surface lipid layer. Determination of the E/M intensity ratio as a function of pyrene lipid concentration in LDL showed a linear relationship for the pyrenyl hexanoate and octanoate species, whereas a slope discontinuity was observed for the lipids containing a longer pyrenyl chain. These data suggest that two lipid domains with distinct properties exist in the surface layer and secondly, pyrenyl lipids partition between these domains in a chainlength-dependent manner. This is consistent with measurement of the tryptophan to pyrene energy transfer efficiency vs. pyrenyl lipid concentration, which showed a biphasic relationship for the long-chain pyrenyl lipids. These measurements further indicate that two surface lipid domains correspond to the protein-lipid boundary and the bulk lipid phase, respectively. The fact that relatively small changes in chainlength have a marked influence on the partitioning of pyrenyl lipids between the boundary and the bulk phase suggests also that native phospholipid species may not be randomly distributed in the surface lipid layer of LDL.

Fluorescence imaging of pyrene-labeled lipids in living cells.

Microscopic imaging of fluorescent lipid derivatives is a powerful tool to study membrane organization and lipid trafficking but it is complicated by cellular autofluorescence background and photobleaching of the fluorophore as well as by the difficulty to selectively image membranes stacked on top of each other. Here we describe protocols that strongly alleviate such problems when pyrene-labeled lipids are being used. First, photobleaching of these lipids is virtually eliminated when oxygen is depleted from the medium by using a gentle and simple enzymatic method. Second, an image practically free of cellular autofluorescence contribution can be obtained simply by subtracting from the pyrene image the background image obtained at a slightly different excitation wavelength. This type of background subtraction more properly accounts for the typically uneven distribution of cellular background fluorescence than other, commonly used methods. Third, it is possible to selectively image the pyrene lipids in the plasma membrane by using plasma membrane-specific quencher trinitrophenyl lysophosphatidylethanolamine and image subtraction. Importantly, either the outer or the inner leaflet can be selectively imaged by labeling the cells with pyrene phosphatidylcholine or phosphatidylserine, respectively. These protocols should be of considerable help when studying organization of the plasma membrane or intracellular lipid trafficking.

Intramitochondrial distribution and transport of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine. Application of pyrene-labeled species.

To investigate the mechanism of intramitochondrial translocation of phosphatidylserine and its decarboxylation product, phosphatidylethanolamine, the distribution of these lipids between the outer (OM) and inner (IM) mitochondrial membranes, as well as their transversal and lateral distribution in OM were studied. Fluorescent, pyrenyl derivatives of phosphatidylserine (PyrxPS) and phosphatidylethanolamine (PyrxPE) species were employed because they allow: (i), direct monitoring of PS (and PE) loading to the mitochondria; (ii) assay of PS decarboxylation by high-performance liquid chromatography with fluorescence detection and (iii), determination of the lateral distributions of PS and PE within the mitochondrial membranes. All PyrxPS species tested were efficiently decarboxylated by the solubilized decarboxylase and thus the distribution of the endogenous PE could be also studied. When the PyrxPS species were loaded to isolated mitochondria very little, if any, of the loaded PyrxPS or of the PyrxPE product was found in IM independent of the time and temperature of incubation, strongly suggesting that these lipids either never enter IM or their residence there is only transient. When mitochondria preloaded with Pyr4PS were incubated with an excess of acceptor vesicles in the presence of the lipid transfer protein, 80% of Pyr4PS and 30-40% of the Pyr4PE product were transported to the acceptor vesicles, indicating that at least corresponding fractions of these lipid were located in, or were in rapid equilibrium with the outer leaflet of OM. Since the decarboxylase is located in the inner membrane, these results signify that both PS and PE must be able to move readily across OM. Determination of the excimer to monomer ratio as the function of pyrenyl lipid concentration in mitochondria (i.e., OM) gave parallel results for PyrxPS and -PE species suggesting the lateral distribution of PS and PE in OM is similar and thus there is no specific enrichment of PS to the contact sites. To investigate the mechanism of PS transport from the outer leaflet to the decarboxylation site, the influence of PyrxPS hydrophobicity, i.e., pyrenylacyl chain length, on the rate of decarboxylation was determined. The variation of the length of the pyrenyl acyl chain from 4 to 12 carbons did not significantly affect the rate of PyrxPS decarboxylation in intact mitochondria, indicating that the transport of PS from the outer leaflet of OM to the site of decarboxylation takes place by lateral diffusion rather than by spontaneous or protein-mediated transport. The implications of these findings on the mechanism of intramitochondrial transport of PS and PE are discussed in terms of alternative models.

Phosphatidylinositol transfer protein from bovine brain. Substrate specificity and membrane binding properties.

A recently developed fluorimetric transfer assay (Somerharju, P., Brockerhoff, H. and Wirtz, K.W.A. (1981) Biochim. Biophys. Acta 649, 521-528) has been applied to study the substrate specificity and membrane binding of the phosphatidylinositol-transfer protein from bovine brain. The substrate specificity was investigated by measuring the rate of transfer, either directly or indirectly, for a series of phosphatidylinositol analogues which included phosphatidic acid, phosphatidylglycerol as well as three lipids obtained from yeast phosphatidylinositol by partial periodate oxidation and subsequent borohydride reduction. Phosphatidylglycerol and the oxidation products of phosphatidylinositol were transferred at about one tenth of the rate observed for phosphatidylinositol while phosphatidic acid was not transferred. It is concluded that an intact inositol moiety favours the formation of the putative transfer protein-phosphatidylinositol complex. In addition to phosphatidylinositol, the transfer protein also transfers phosphatidylcholine. In order to obtain information on the possible occurrence of two sites of interaction, vesicles consisting of either pure 1-acyl-2-parinaroylphosphatidylinositol or 1-acyl-2-parinaroylphosphatidylcholine were titrated with the protein. Binding of labeled phospholipid to the protein was represented by an increase of lipid fluorescence and found to be much more efficient for phosphatidylinositol than for phosphatidylcholine. This is interpreted to indicate that the protein contains an endogenous phosphatidylinositol molecule which can be easily replaced by exogenous phosphatidylinositol but not by phosphatidylcholine, a lipid with a lower affinity for this protein. Thus the binding sites for the two phospholipids are mutually exclusive, i.e. phosphatidylinositol and phosphatidylcholine cannot be bound to the protein simultaneously. Finally, the effect of acidic phospholipids on the transfer protein activity was studied either by varying the content of phosphatidic acid in the acceptor vesicles or by adding vesicles of pure acidic phospholipids to the normal assay system. The latter vesicles consisted of either phosphatidic acid, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol or cardiolipin. In both instances the transfer protein activity was inhibited, obviously through the enhanced association of the protein with the negatively charged vesicles. These findings strongly suggest that relatively nonspecific ionic forces rather than specific protein-phospholipid headgroup interactions contribute to the association of the phosphatidylinositol-transfer protein with membranes.

Lateral organisation of membrane lipids. The superlattice view.

Most biological membranes are extremely complex structures consisting of hundreds or even thousands of different lipid and protein molecules. The prevailing view regarding the organisation of these membranes is based on the fluid-mosaic model proposed by Singer and Nicholson in 1972. According to this model, phospholipids together with some other lipids form a fluid bilayer in which these lipids are diffusing very rapidly laterally. The idea of rapid lateral diffusion implies that, in general, the different lipid species would be randomly distributed in the plain of the membrane. However, there are recent data indicating that the components tend to adopt regular (superlattice-like) distributions in fluid, mixed bilayers. Based on this, a superlattice model of membranes has been proposed. This superlattice model is intriguing because it allows only a limited certain number of 'critical' compositions. These critical compositions could play a key role in the regulation of the lipid compositions of biological membranes. Furthermore, such putative critical compositions could explain how compositionally distinct organelles can exist despite of rapid inter-organelle membrane traffic. In this review, these intriguing predictions are discussed along with the basic principles of the model and the evidence supporting it.

A new fluorimetric method to measure protein-catalyzed phospholipid transfer using 1-acyl-2-parinaroylphosphatidylcholine.

A new, simple and versatile method to measure phospholipid transfer has been developed, based on the use of a fluorescent phospholipid derivative, 1-acyl-2-parinaroylphosphatidylcholine. Vesicles prepared of this phospholipid show a low level of fluorescence due to interaction between the fluorescent groups. When phospholipid transfer protein and vesicles consisting of non-labeled phosphatidylcholine are added the protein catalyzes an exchange of phosphatidylcholine between the labeled donor and non-labeled acceptor vesicles. The insertion of labeled phosphatidylcholine into the non-labeled vesicles is accompanied by an increase in fluorescence due to abolishment of self-quenching. The initial rate of fluorescence enhancement was found to be proportional to the amount of transfer protein added. This assay was applied to determine the effect of membrane phospholipid composition on the activity of the phosphatidylcholine-, phosphatidylinositol- and non-specific phospholipid transfer proteins. Using acceptor vesicles of egg phosphatidylcholine and various amounts of phosphatidic acid it was observed that the rate of phosphatidylcholine transfer was either stimulated, inhibited or unaffected by increased negative charge depending on the donor to acceptor ratio and the protein used. In another set of experiments acceptor vesicles were prepared of phosphatidylcholine analogues in which the ester bonds were replaced with ether bonds or carbon-carbon bonds. Assuming that only a strictly coupled exchange between phosphatidylcholine and analogues gives rise to the observed fluorescence increase, orders of substrate preference should be established for the phosphatidylcholine- and phosphatidylinositol transfer proteins.

Stereoconfiguration of bisphosphatidic and semilysobisphosphatidic acids from cultured hamster fibroblasts (BHK cells).

Monolayers of hamster fibroblasts (BHK cells) were incubated in Eagle's minimal essential medium under conditions where an increase in the levels of all cellular bisphosphatidic acids takes place. Bisphosphatidic acid and semilysobisphosphatidic were isolated from these cells and subjected to strong alkaline hydrolysis. Stereochemical analysis of the hydrolysis products revealed that the majority of the molecules of both lipids are derivatives of sn-1-glycerophospho-sn-1'-glycerol, the structure previously found to be the "backbone" of lysobisphosphatidic acid, (bis(monoacylglycerol)phosphate) from BHK cells and other sources. This finding suggests a close metabolic relationship between the three bisphosphatidic acid derivatives of BHK cells.

On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels.

The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC). In this study we have determined the affinity of PI-transfer protein for PI relative to that for PC by measuring the binding of the fluorescent pyrene-labeled analogs of these phospholipids. From competition binding experiments it was estimated that the transfer protein has a 16-fold higher affinity for PI than for PC. This relative affinity together with the relative abundance of PI and PC, determines what proportion of the protein contains PI (e.g. 65% of the PI-transfer protein in the case of bovine brain). From measuring lipid transfer between donor vesicles consisting of equimolar amounts of PC and PI, and an excess of acceptor vesicles consisting of various ratios of PC and PI, we have observed that the relative rates of the PI-transfer protein-mediated transfer of PI and PC varies between 5 and 20. Kinetic analysis has indicated that PI-transfer protein carrying a PI molecule has different kinetic properties than the PI-transfer protein carrying a PC molecule. It will be discussed that because of the dual specificity, PI-transfer protein is ideally suited for maintaining PI levels in intracellular membranes.

Acyl chain and headgroup specificity of human plasma phospholipid transfer protein.

Phospholipid transfer protein (PLTP) is a plasma protein with two reported in vitro activities: transfer of phospholipids and modulation of HDL particle size. The mechanism of PLTP-mediated phospholipid transfer was studied by determining the acyl chain and headgroup specificity and comparing the results with those obtained with the non-specific lipid transfer protein (ns-LTP), a previously characterised intracellular transfer protein. To verify the results obtained with purified plasma PLTP, recombinant PLTP produced in COS-1 cells was used. The transfer rates were determined by monitoring the transfer of fluorescent, pyrene-labeled phospholipids from quenched donor phospholipid vesicles to HDL3 particles. When the length of the pyrene-labeled acyl chain was varied from 6 to 14 carbons, a fairly monotonous decrease in the transfer rate was observed. No difference in rate was observed for the isomers having the pyrene-labeled and unlabeled acyl chains in reversed positions. PLTP mediated equally the transfer of the various headgroup derivatives except phosphatidylethanolamine (PE), which was transferred 2-3-fold more slowly. In all experiments the plasma and recombinant PLTP behaved identically. The specificity patterns observed for PLTP and ns-LTP were very similar. No PLTP-phospholipid intermediate could be observed, indicating that PLTP, like ns-LTP, does not form a tight complex with the lipid substrate and may thus mediate the transfer of phospholipid via another, yet unspecified mechanism.

Oxidative modification of HDL3 in vitro and its effect on PLTP-mediated phospholipid transfer.

The oxidation of HDL3 by Cu(II) and its effect on the ability of these particles to act as phospholipid acceptors in human plasma phospholipid transfer protein (PLTP)-mediated lipid transfer were investigated. Oxidation of HDL3 was monitored by measuring the following parameters: (i) formation of conjugated dienes, (ii) production of thiobarbituric acid reactive substances (TBARS), (iii) decrease in reactive lysine and (iv) tryptophan residues, (v) change in particle charge and (vi) diameter, and (vii) oligomerisation of apoA-I and apoA-II. Formation of conjugated dienes was the parameter responding to the oxidative treatment with the fastest kinetics. The appearance of TBARS and modification of apolipoprotein tryptophan residues were detected simultaneously but required higher Cu(II) concentrations for maximal kinetics. Cross-linking of the major protein constituents of HDL3, apoA-I and apoA-II, represented later steps of the oxidation process. Further, the oxidative modification was accompanied by a progressive change in HDL3 particle charge and a minor increase in particle diameter. PLTP-mediated phospholipid transfer to the oxidized particles was investigated using an assay measuring the transfer of fluorescent, pyrene-labeled PC. The transfer was significantly inhibited, but only after extensive modification of the HDL proteins, suggesting that the HDL oxidative modifications occurring in vivo do not essentially impair its phospholipid acceptor function. A similar but less pronounced inhibition was observed when two other phospholipid transfer proteins, the nonspecific lipid transfer protein (ns-LTP) and the phosphatidylcholine transfer protein (PC-TP), were studied in parallel. This indicates that the inhibition was partly due to unspecific effects of the modification on acceptor particle surface properties, but included an aspect specific for PLTP.

Human postheparin plasma hepatic lipase activity against triacylglycerol and phospholipid substrates.

Recent evidence has suggested that the major physiological substrate of the heparin-releasable (postheparin plasma) hepatic lipase is HDL2-phospholipid. However, all the current assay methods for this enzyme are based on the use of triacylglycerol substrate. Even though both lipolytic activities of hepatic lipase are likely to be due to a single enzyme it is possible that the use of unphysiological lipid as a substrate may give misleading results. Therefore we did parallel assays of the activity of postheparin plasma hepatic lipase using triacylglycerol, monoacylglycerol and phospholipid substrates. The correlation coefficients between the three lipolytic activities were 0.95-0.98, indicating that identical results are obtained using any of these three lipids in the assay of postheparin plasma hepatic lipase.

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine.

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidylethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.

Intramolecular excimer formation of pyrene-labeled lipids in lamellar and inverted hexagonal phases of lipid mixtures containing unsaturated phosphatidylethanolamine.

The rates of intramolecular excimer formation of di(1'-pyrenemyristoyl)phosphatidylcholine (dipyPC) in dioleoylphosphatidyl-ethanolamine (DOPE), egg PE/diolein (DG) and dilinoleoyl-PE (DLPE)/1-palmitoyl-2-oleoyl-PC (POPC) were studied at different temperatures and lipid compositions. Both the excimer-to-monomer intensity ratio and the excimer association rate constant were employed to quantify the rate of excimer formation. The latter was calculated from the measured monomer fluorescence lifetime of dipyPC. We observed that the rate of excimer formation was sensitive to either the temperature-induced or lipid composition-induced lamellar-to-inverted hexagonal phase transition of the above lipid systems. As the lipids entered the inverted hexagonal phase, the rate of excimer formation increased at the temperature-induced phase transition for DOPE, but decreased at the composition-induced phase transition for both TPE/DG and DLPE/POPC systems by increasing the DG% and decreasing the PC%, respectively. We conclude that the rate of intramolecular excimer formation of dipyPC in the non-lamellar phase is influenced both by the intra-lipid free volume of the hydrocarbon region and the intra-rotational dynamics of the two lipid acyl chains.

The surface lipid layer of human low density lipoprotein probed by dipyrenyl phospholipids.

Properties of the surface lipid-protein layer of human low density lipoprotein (LDL) have been studied with fluorescent phosphatidylcholine analogues containing a pyrenyl fatty acid of variable length at both sn-1 and sn-2 position of the glycerol moiety. Only intramolecular excimer formation takes place at low concentrations, as indicated by the independence of the ratio of excimer to monomer fluorescence intensities (E/M) on the amount of the incorporated dipyrenyl phospholipid. The E/M parameter which depends on the fluidity of the probe's environment were measured for a series of dipyrenyl phospholipids in three systems, i.e. in LDL, LDL-like lipid particles (LDp) and small unilamellar phosphatidylcholine/sphingomyelin/cholesterol vesicles (SUV). The data indicate that the fluidity of the phospholipid acyl chain region decreases in the order: SUV greater than LDp greater than LDL. This suggests that interactions with both the core lipids and the protein moiety (apoB-100) contribute to the rigidity of the surface lipid layer of LDL. Dipyrenyl phospholipids also detect the thermotropic transition of the core lipids of both LDL and LDp, suggesting that this transition influences the fluidity of the surface lipid layer.