Phospholipid Molar Ratio Research Articles

Lysosomal membrane cholesterol dynamics.

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.

Abnormalities of erythrocyte membrane fluidity, lipid composition, and lipid peroxidation in systemic sclerosis: evidence of free radical-mediated injury.

To elucidate whether oxidative injury occurs in systemic sclerosis (SSc) and whether it affects the erythrocyte membrane (EM) properties. EM fluidity and lipid composition (cholesterol:phospholipid molar ratio [C:PL], fatty acid composition) were studied in 52 patients with SSc and in 53 subjects without SSc (32 with primary Raynaud's phenomenon [RP] and 21 healthy subjects [controls]). Fluidity was measured as the fluorescence anisotropy of the hydrophobic fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene). Lipid peroxidation products were determined as thiobarbituric acid-reactive substances (TBARS). EM fluidity was significantly lower in SSc patients than in primary RP patients and controls (P < 0.001). The EM C:PL molar ratio was significantly higher in SSc patients than in primary RP patients and controls (P < 0.05). Levels of EM polyunsaturated n6 fatty acids (PUFA n6) were significantly lower in SSc patients than in primary RP patients and controls (P < 0.001). TBARS were significantly increased in SSc patients compared with primary RP patients and controls (P < 0.001). Multiple regression analyses indicated that the reduced EM fluidity was partly due to its greater C:PL molar ratio, lower PUFA n6 content, and higher TBARS levels. EM fluidity was lower among patients with nailfold capillary loss (P < 0.001) and digital ischemic ulcers (P < 0.05). EM lipid peroxidation products were higher among patients with pulmonary involvement (bibasal pulmonary fibrosis [P < 0.05] and reduced levels of diffusing capacity for carbon monoxide [P < 0.001]) and among patients who were positive for anti-topoisomerase I antibodies (P < 0.05) or negative for anticentromere antibodies (P < 0.001). Our findings support the idea that oxidative injury occurs in SSc and that, through lipid peroxidation, it induces structural and functional changes of the EM that may contribute to the development of the microvascular abnormalities that are seen in the disease.

Distribution of lipid and protein in human semen fractions

Human semen is formed by the secretions of different glands. We fractionated semen by centrifugation and obtained four main fractions: (a) spermatozoa, (b) material precipitating at 10 000× g, (c) prostasomes (precipitate at 105 000× g), and (d) a soluble fraction. When required, fractions were purified further. We find that most semen protein (about 85%) is in the soluble fraction, 7% in spermatozoa and the remainder is scattered in the other fractions. We compared the electrophoretic pattern of soluble protein with the protein of prostasomes and found marked differences. On the other hand, prostasomes, that comprises only about 3% of total semen protein, contain about 45% of cholesterol and almost 15% of lipid phosphorus with a cholesterol to phospholipid molar ratio greater than 2. On the contrary, phospholipid is largely bound to the fraction containing spermatozoa (about 46% of total lipid phosphorus). This fraction is poor in cholesterol and has a cholesterol to phospholipid molar ratio of about 0.2. The distribution of lipid phosphorus among lipid classes shows some similarity in the soluble fraction and in prostasomes; in both fractions, sphingomyelin is the most abundant phospholipid (about 50%). On the other hand, phosphatidylcholine is the main phospholipid in spermatozoa-enriched fractions (about 35% of total lipid phosphorus). We conclude that the various fractions of seminal plasma obtained by centrifugation differ markedly from each other as to lipid and protein content.

Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis

Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37 degrees C with a unit cell periodicity of 34 A coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binary lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol-phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.

A high-sucrose diet alters the lipid composition and fluidity of liver sinusoidal membranes.

Impaired insulin suppression of hepatic glucose production and accumulation of hepatic triglycerides occur after 1 week on a high-sucrose diet. The purpose of this study was to ascertain whether changes in structural lipids, fatty acid composition and/or fluidity occur after 1 week on a high-sucrose diet, and therefore might contribute to the sucrose-induced impairment in hepatic glucose metabolism. Male Wistar rats (n=28) were fed a purified high starch (68% of energy) diet for a 2-week baseline period. Fourteen animals were then switched to a high sucrose (68% of energy) diet for 1 (n=7) or 5 (n=7) weeks. Analyses were performed on liver sinusoidal membranes (due to this membrane's involvement in nutrient transport) from overnight fasted rats. The degree of saturation of sinusoidal membrane phospholipids and liver triglyceride fatty acids was significantly greater in sucrose vs. starch at 1 and 5 weeks. This resulted in significantly lower sinusoidal membrane fluidity at 1 and 5 weeks in the sucrose group. In contrast, hepatic sinusoidal membrane cholesterol content (0.60+/-0.05 vs. 0.42+/-0.04 micromol/mg protein) and the cholesterol to phospholipid molar ratio (0.66+/-0.04 vs. 0.50+/-0.03) were significantly greater in sucrose vs. starch animals at 5 weeks only. Minimal differences were observed in individual phospholipid species between groups. These data suggest that changes in fatty acid composition and fluidity may contribute to the development of sucrose-induced hepatic insulin resistance.

Changes in the Neuronal Membranes of Mice Related to Steroid Hormone Influences

Changes in the biochemical composition of synaptosomal plasma membranes (SPM) isolated from mouse brains have been measured. The protein, phospholipid, and cholesterol contents all increased over the first 30 days of postnatal life, with the cholesterol to phospholipid molar ratio (one of the major determinants of lipid fluidity) also increasing in direct relation to the decrease in lipid fluidity. The fatty acid composition of SPM also changes with the increase in 18:0, and the decrease in 18:2, 18:3, and 22:4, in keeping with the increase in membrane order. Steroid hormones alter lipid fluidity to a greater degree in fluid membranes, indicating that the nongenomic effects of steroids will be most prevalent in membranes during the early prenatal period and for the first days following birth. The potential effects of xenobiotics on membrane fluidity are also discussed.

Comparison of the interaction of doxorubicin, daunorubicin, idarubicin and idarubicinol with large unilamellar vesicles: Circular dichroism study

Doxorubicin, daunorubicin and other anthracycline antibiotics constitute one of the most important groups of drugs used today in cancer chemotherapy. The details of the drug interactions with membranes are of particular importance in the understanding of their kinetics of passive diffusion through the membrane which is itself basic in the context of multidrug resistance (MDR) of cancer cells. Anthracyclines are amphiphilic molecules possessing dihydroxyanthraquinone ring system which is neutral under the physiological conditions. Their lipophilicity depends on the substituents. The amino sugar moiety bears the positive electrostatic charge localised at the protonated amino nitrogen. The four anthracyclines used in this study doxorubicin, daunorubicin, idarubicin and idarubicinol (an idarubicin metabolite readily formed inside the cells) have the same amino sugar moiety, daunosamine, with p K a of 8.4. Thus, all drugs studied will exhibit very similar electrostatic interactions with membranes, while the major differences in overall drug-membrane behaviour will result from their hydrophobic features. Circular dichroism (CD) spectroscopy was used to understand more precisely the conformational aspects of the drug–membrane systems. Large unilamellar vesicles (LUV) consisting of phosphatidylcholine, phosphatidic acid (PA) and cholesterol, were used. The anthracycline–LUV interactions depend on the molar ratio of phospholipids per drug. At low molar ratios drug:PA, these interactions depend also on the anthracycline lipophilicity. Thus, both doxorubicin and daunorubicin bind to membranes as monomers and their CD signal in the visible is positive. However, doxorubicin with its very low lipophilicity binds to the LUV through electrostatic interactions, with the dihydroxyanthraquinone moiety being in the aqueous phase, while daunorubicin, which is more lipophilic is unable to bind only through electrostatic interactions and actually the hydrophobic interactions are the only detected. The highly hydrophobic idarubicin, forms within the bilayer a rather complex entity involving 2–3 molecules of idarubicin associated in the right-handed conformation, one cholesterol molecule and also molecule(s) of phosphatidic acid, as this special oligomeric species is not detected in the absence of negatively-charged phospholipids. Idarubicinol differs from idarubicin with CH(13)–OH instead of C(13)O and its interactions with LUV are distinctly different. Its CD signal in the visible becomes negative and no self associations of the molecule within the bilayer could be detected. The variation of the sign of the Cotton effect (positive to negative) may derive from the changes in the C(6a)–C(7)–O(7)–C(1′) dihedral angle. It is noteworthy that C(13)–OH group, which strongly favours formation of the dimeric species in aqueous solutions when compared to idarubicin prevent association inside the LUV bilayer. At high ratios of phospholipids per drug all of them are embedded within the bilayer as monomer.

Subsolubilizing alterations caused by alkyl glucosides in phosphatidylcholine liposomes

The subsolubilizing alterations caused by a series of alkyl glucosides (alkyl chain lengths ranging from C 8 to C 12) in unilamellar phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios ( R e) and the normalized bilayer/aqueous phase partition coefficients ( K) were determined by monitoring the increase in the fluorescence intensity of liposome suspensions due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations was always lower than the critical micelle concentration (CMC) of the surfactant tested we may assume that the surfactant–liposome interactions were mainly ruled by the action of surfactant monomers. In general terms, the decrease in the surfactant alkyl chain length (or the rise in the surfactant CMC) resulted in an increase in the ability of these surfactants to alter the permeability of liposomes and, inversely, in an abrupt decrease in their affinity with these bilayers structures. The overall balance of these opposite tendencies shows that at the two interaction levels studied (50 and 100% of CF release) the nonyl and the octyl glucoside showed, respectively, the highest ability to alter the release of the CF trapped in bilayers (lowest R e values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values).

Reduction of sperm cholesterol:phospholipid ratio is a possible mechanism for enhancement of human sperm binding to the zona pellucida following incubation with phosphatidylcholine liposomes.

TEST (TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid) and Tris) yolk buffer (TYB) has recently been shown to improve the binding capacity of spermatozoa to zona pellucidae. The present study had two objectives: 1) to elucidate which component(s) of TYB dominates this effect and 2) to define the responsible mechanism. Sperm samples obtained from subfertile men were incubated for 2 h in either TYB or media containing egg yolk lipoproteins or phospholipids. After incubation, sperm binding was tested by the hemizona assay. Yolk lipoprotein-treated spermatozoa bound hemizonae with efficiency equal to that of the spermatozoa incubated in control medium. Conversely, incubation of spermatozoa in media containing either TYB, yolk-phospholipids, or pure phosphatidylcholine (PC) resulted in a 2- to 3-fold increased binding capacity (p < 0.01). A close correlation was found between the effect of yolk-phospholipids and TYB on the binding capacity of the same sperm samples, compared to spermatozoa incubated in control medium. Incubation of spermatozoa in yolk phospholipid medium caused a dose-dependent increase of sperm binding capacity (p < 0.05). Treatment of sperm samples with 1 mg/ml or more of purified PC preparation also resulted in a reduction of the sperm cholesterol:phospholipid molar ratio. Significant correlations between the effects of the treatments on sperm cholesterol: phospholipid molar ratio and sperm binding were obtained with yolk-phospholipids (r = -0.55) or 1 mg/ml purified PC (r = -0.61). We conclude that 1) the enhanced binding capacity of human spermatozoa following TYB treatment is probably due to yolk-phospholipids, mainly egg yolk PC; and 2) it appears that the enhanced binding capacity of human spermatozoa following treatment with egg yolk-containing media may be a result of the reduction of the cholesterol:phospholipid molar ratio in the sperm cells.

Lipid Composition and Fatty Acid Profiles of Myelin and Synaptosomal Membranes of Rat Brain in Response to the Consumption of Different Fats

Experiments were performed to ascertain the effect of dietary fats like safflower, mustard, peanut, and coconut oil, which differ widely in the essential fatty acid components on the distribution patterns of specific lipids such as cholesterol, phospholipids, and cerebrosides and the fatty acid profile of the latter two lipid fractions obtained from myelin and synaptosomal membranes of three anatomical regions of the rat brain (cerebrum, cerebellum, and brain stem). Weanling male CFY rats received diets adequate in all essential nutrients but varied with respect to the nature of dietary fat. The fat was fed at the 20% level in the diet for 16 weeks. Myelin and synaptosomal membranes from cerebrum, cerebellum, and brain stem of these rat brains were prepared using discontinuous sucrose density gradient ultracentrifugation. Cholesterol to phospholipid molar ratio remained constant in the synaptosomal membranes in cerebrum and brainstem regions irrespective of the dietary fat treatment, except in the cerebellar region of coconut and mustard oil-fed animals, which showed significantly higher ratio. The cerebroside content of myelin membranes obtained from cerebra and cerebella of these animals were significantly lower compared with those of groundnut and safflower oil-fed rats. Fatty acid compositions of myelin and cerebroside fractions of these membranes were determined. Mustard oil feeding resulted in the enrichment of synaptosomal phospholipid fraction with docosahexaenoic (22:6 n-3) acid (DHA) in the cerebral and cerebellar regions. On the contrary, myelin phospholipid fractions of these regions of mustard oil-fed group were characterized by a lower degree of unsaturation. Lignoceric (24:0) and nervonic (24:1 n-9) acids, the marker fatty acids of myelin, were significantly lower in the brain regions of coconut and mustard oil-fed animals. Results indicate that the brain can adapt to essential fatty acid deficiency in response to coconut oil feeding by retaining high concentrations of polyunsaturated fatty acid in its membranes. On the other hand, dietary fats rich in linoleic (18:2 n-6) acid (safflower) or linolenic acid (18:3 n-3) (mustard oil) significantly elevated the polyunsaturated fatty acid content. In addition, they also altered the long chain fatty acids (like lignoceric and nervonic acids) of a stable membrane, like myelin.

Vesicle to micelle phase transitions involved in the interaction of sodium cholate with phosphatidylcholine liposomes

The interaction of sodium cholate surfactant with phosphatidylcholine unilamellar liposomes was investigated. Alterations in bilayer permeability were detected as a change in 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous medium and bilayer solubilization as a decrease in the static light-scattered by liposome suspensions. The value corresponding to the effective surfactant to phospholipid molar ratio parameter ( Re) for each interaction step investigated progressively increased as the surfactant concentration rose, this rise being more pronounced during bilayer solubilization. At the subsolubilizing level, a maximum bilayer/water surfactant partitioning ( K) was reached at 30% of CF release, correlated with the saturation of the outer vesicle leaflet, followed by an abrupt decrease in K up to the complete saturation of these bilayer structures ( Re SAT). At the solubilizing level, the K parameter also drastically increased up to the complete bilayer solubilization ( Re SOL). The fact that the free surfactant concentration at subsolubilizing and solubilizing levels showed values respectively lower than and similar to its critical micelle concentration (CMC) indicates that permeability alterations and bilayer solubilization were determined respectively by the action of surfactant monomer and by the formation of mixed micelles.

Dietary Oxidized Linoleic Acid Modifies Lipid Composition of Rat Liver Microsomes and Increases Their Fluidity

The effect of dietary oxidized oil on the lipid composition, fluidity and function of rat liver microsomes was studied. Male growing rats were fed diets containing 10 g/100 g of a fresh (control) or oxidized (experimental) linoleic acid–rich preparation for 4 wk. High levels of fluorescent compounds and of thiobarbituric acid reactive substances indicated the occurrence of substantial lipid peroxidation in the microsomes of the experimental rats. The fluidity of the liver microsomes derived from rats fed the experimental diet was significantly higher than that of the membranes of the controls. This was due to profound differences in lipid composition of the liver microsomes, namely, a lower cholesterol to phospholipid molar ratio and a greater arachidonic acid content in the phospholipids of the rats fed the experimental diet. The fluidity differences were accompanied by greater activity of the microsomal enzymes, aldehyde dehydrogenase and NADPH cytocrome C reductase. The study demonstrated that ingestion of oxidized lipids caused profound alterations in membrane composition, fluidity and function. These alterations are likely to be associated with an enhanced cholesterol turnover, as indicated by the greater cholesterol excretion observed for the experimental rats.

Evidence That the Initial Up-regulation of Phosphatidylcholine Biosynthesis in Free Cholesterol-loaded Macrophages Is an Adaptive Response That Prevents Cholesterol-induced Cellular Necrosis: PROPOSED ROLE OF AN EVENTUAL FAILURE OF THIS RESPONSE IN FOAM CELL NECROSIS IN ADVANCED ATHEROSCLEROSIS

Macrophages in atherosclerotic lesions accumulate free cholesterol (FC) as well as cholesteryl ester and appear to have high rates of phospholipid (PL) synthesis and increased PL mass. Previous short term (i.e. </=24 h) studies with cultured macrophages have shown that these cells respond to FC loading by up-regulating phosphatidylcholine biosynthesis. We propose that this response is adaptive by keeping the FC:PL ratio in the macrophages from reaching toxic levels. We further propose that one cause of macrophage necrosis, a prominent and important event in atherosclerosis, is an eventual decrease of this adaptive response. To explore these ideas, cultured macrophages were loaded with FC for up to 4 days and assayed for phosphatidylcholine biosynthesis, FC and PL mass, and cytotoxicity. For the first 24 h, cellular phosphatidylcholine biosynthesis and FC and PL mass increased 3-4-fold, and thus the FC:PL molar ratio was prevented from reaching very high levels; at this point, there were no overt signs of cytotoxicity. Over the next 24-48 h, however, phosphatidylcholine biosynthesis, and then phosphatidylcholine mass, began to decrease. Initially, the macrophages remained healthy and continued to accumulate FC, but eventually these macrophages, but not unloaded macrophages, became necrotic (swollen organelles and disrupted membranes). Lipoprotein dose studies indicated a close relationship between the onset of macrophage necrosis and the FC:PL ratio. To test further the causal nature of these relationships, cellular FC and PL mass were independently manipulated by using high density lipoprotein3 (HDL3) to decrease cellular FC and choline depletion to decrease cellular PC. As predicted by our hypotheses, HDL3 protected FC-loaded macrophages from necrosis, whereas choline depletion accelerated cytotoxic changes. These findings support the idea that the initial increase in phosphatidylcholine biosynthesis in FC-loaded macrophages is an adaptive response that prevents cholesterol-induced macrophage necrosis. We propose that an eventual failure of the PL response in foam cells may represent one cause of macrophage necrosis in advanced atherosclerotic lesions.

Membrane cholesterol alters gallbladder muscle contractility in prairie dogs.

The cause and effect relationship between membrane cholesterol and gallbladder muscle contractility was examined by altering membrane cholesterol to phospholipid mole ratio using cholesterol-rich or cholesterol-free liposomes. Gallbladder single muscle cells, from prairie dogs that were fed either a regular or high-cholesterol (1.2%) diet, were isolated enzymatically with collagenase. Plasma membranes of gallbladder muscle were purified in sucrose gradient. Cholesterol was measured using the cholesterol oxidase method. Phospholipids were measured with the method of G.R. Bartlett (J. Biol. Chem. 234: 466-468, 1959). The results of this experiment are 1) after high-cholesterol feeding, cholesterol contents and cholesterol/ phospholipid mole ratio in plasma membranes of gallbladder muscle increased 90%, and muscle cell contraction in response to cholecystokinin octapeptide decreased 58%; 2) similar changes were observed when normal gallbladder muscle cells were incubated with cholesterol-rich liposomes for 2 h; and 3) the changes induced either in vivo or in vitro were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes for 2-6 h. We conclude that gallbladder muscle may incorporate excess cholesterol into its plasma membrane when exposed to a cholesterol-rich environment, that excess membrane cholesterol impairs muscle contractility, and that these changes appear to be reversible.

Role of membrane fluidity in Epstein Barr virus (EBV) infectivity on Akata cell line

Infection of Epstein Barr virus (EBV) to its host cells is initiated by the attachment of the glycoprotein gp 350/220 to the CR2 molecule. We used the sensitivity at the polar environment of the fluorescent probe Laurdan to study the membrane viscosity distribution from single leaving cells on two lymphoid cell lines Raji and Akata. Lipid analysis on both cells line demonstrated a lower cholesterol to phospholipid molar ratio on Akata than Raji cells. Cell fluidity analysis by Laurdan generalized emission polarization (GP) or by DPH polarization, indicated that membrane viscosity of Akata was lower than Raji cells. This difference was correlated to the increased susceptibility of Akata cells in expressing EBV early antigens (EA) after EBV superinfection.

Laurdan fluorescence spectroscopy reveals a single liquid-crystalline lipid phase and lack of thermotropic phase transitions in the plasma membrane of living human sperm

Membrane lipid phase(s), phase coexistence, and thermotropic phase transitions have been investigated in viable human spermatozoa using Laurdan fluorescence spectroscopy. Generalized polarization (GP) values derived from Laurdan excitation and emission spectra confirm that the sperm plasma membrane is a low polar, highly rigid (liquid-ordered) structure, and give evidence that, in the range from 10°C to 42°C, membrane lipids are in a single liquid-crystalline phase. The absence of phase transitions in the same thermal range argues against the hypothesis that the lipid domains previously detected on the sperm surface are produced by lipid lateral phase separation. The above findings are likely accounted for by the high cholesterol to phospholipid molar ratio found in the human sperm membrane. This is the first time that membrane lipid phase and polarity have been detected and quantified in living mammalian spermatozoa.

Evidence for phospholipid bilayer formation in solid lipid nanoparticles formulated with phospholipid and triglyceride.

Solid lipid nanoparticles (SLN) are comprised of a high-melting point triglyceride (TG) core with a phospholipid (PL) coating. This study has investigated the possible formation of multiple PL bilayers on the TG core of SLN's as a function of increasing the PL:TG molar ratio. Trilaurin (TL) was used as the SLN core. Dipalmitoylphos-phatdylcholine (DPPC) or a mixture of DPPC and dimyristoylphosphatidylglycerol (DMPG) were used to produce neutral and negatively charged SLN's. The volume of aqueous phase associated with the PL was determined using calcein and 6-carboxyfluorescein (6-CF) as hydrophilic markers incorporated during the preparation of the SLN's. The diameter of the SLN's decreased as the molar ratio of PL to TL was increased, until a PL:TL ratio of 0.15 was reached. After this point the diameter was not affected by further increases in the molar ratio. The experimental amount of PL required to prepare SLN's was significantly higher than the theoretical amount required to form a single monolayer on the surface. The aqueous volume associated with the PL was increased with increasing PL:TL molar ratios. The results obtained suggest that the formation of multiple PL bilayers is probable in SLN's prepared with a high molar ratio of PL to TL. The volume of the aqueous phase between the PL-bilayers, estimated from the amount of the hydrosoluble markers trapped in this phase, provides an indication of the relative number of bilayers at different PL:TL ratios.

Deoxycholic acid influences cholesterol solubilization and microcrystal nucleation time in gallbladder bile

Little is known about the effects of biliary deoxycholic acid on the partitioning of biliary cholesterol between vesicles and micelles and on the rate of nucleation of cholesterol microcrystals, key steps in gallstone formation. Therefore, 43 samples of fresh gallbladder bile were obtained from a heterogeneous group of patients with and without stones. Univariate and multivariate analyses were then applied to determine the inter-relationships between biliary cholesterol saturation, total lipid concentration, and bile acid species and (1) the distribution of biliary cholesterol between vesicles and micelles and (2) the cholesterol microcrystal nucleation time. The percentage of deoxycholic acid in bile was shown to be linearly related to the cholesterol saturation index ( r = .54; P < .001), the vesicular cholesterol:phospholipid molar ratio ( r = .53; P < .001), and the molar concentration of cholesterol in the vesicles ( r = .59; P < .001). The mean proportion of biliary deoxycholic acid conjugates was also greater in patients with rapid nucleation times (23.4 ± SEM 1.1%) than in those with slow nucleation times (17.3 ± 1.9%; P < .05). As total bile lipid concentration increased, the proportion of total biliary cholesterol in vesicles decreased ( r = .53; P < .001), whereas the molar concentration of vesicular cholesterol increased ( r = .42, P < .01). The cholesterol saturation indices, total bile lipid concentration, and proportion of biliary deoxycholate were independent determinants of the molar concentration of cholesterol in vesicles. We conclude that relative increases in the percentage of deoxycholic acid and in bile lipid concentration, favor the partitioning of cholesterol into vesicles. In turn, this leads to an increase in the vesicular cholesterol : phospholipid molar ratio and thus to a decrease in the cholesterol microcrystal nucleation time.

Deoxycholic acid influences cholesterol solubilization and microcrystal nucleation time in gallbladder bile

Little is known about the effects of biliary deoxycholic acid on the partitioning of biliary cholesterol between vesicles and micelles and on the rate of nucleation of cholesterol microcrystals, key steps in gallstone formation. Therefore, 43 samples of fresh gallbladder bile were obtained from a heterogeneous group of patients with and without stones. Univariate and multivariate analyses were then applied to determine the inter-relationships between biliary cholesterol saturation, total lipid concentration, and bile acid species and (1) the distribution of biliary cholesterol between vesicles and micelles and (2) the cholesterol microcrystal nucleation time. The percentage of deoxycholic acid in bile was shown to be linearly related to the cholesterol saturation index (r = .54; P < .001), the vesicular cholesterol:phospholipid molar ratio (r = .53; P < .001), and the molar concentration of cholesterol in the vesicles (r = .59; P < .001). The mean proportion of biliary deoxycholic acid conjugates was also greater in patients with rapid nucleation times (23.4 +/- SEM 1.1%) than in those with slow nucleation times (17.3 +/- 1.9%; P < .05). As total bile lipid concentration increased, the proportion of total biliary cholesterol in vesicles decreased (r = .53; P < .001), whereas the molar concentration of vesicular cholesterol increased (r = .42, P < .01). The cholesterol saturation indices, total bile lipid concentration, and proportion of biliary deoxycholate were independent determinants of the molar concentration of cholesterol in vesicles. We conclude that relative increases in the percentage of deoxycholic acid and in bile lipid concentration, favor the partitioning of cholesterol into vesicles. In turn, this leads to an increase in the vesicular cholesterol:phospholipid molar ratio and thus to a decrease in the cholesterol microcrystal nucleation time.

Phospholipid Degradation Is Induced by Heat in α-Tocopherol-Enriched Eggs

A comparative study was undertaken to determine the effect of an α-tocopherol- and iodine-enriched laying diet on the phospholipid profile of egg yolk. In addition to the comparative study between the experimental and control eggs, experiments were conducted to determine the effects of heating on the phospholipid profile and the comparative molar ratios of cholesterol and total phospholipid. The phospholipid composition determined for frozen egg yolk samples showed no differences for the major components of phosphatidyl-choline and phosphatidylethanolamine between the control and experimental diet group. In control eggs, exposure to boiling water produced time-related elevations in the concentration of lyso-phosphatidylcholine. A similar heat-related elevation in lyso-phosphatidylethanolamine was observed in both groups after 10 min. A time shift was observed in the heat susceptibilities of the experimental diet group. The control egg yolks hardened more quickly when exposed to heat. The results suggest protection against oxidative degradation of phospholipids and possible inhibition of phospholipases A2 and C, which may result from the elevated level of α-tocopherol in the experimental egg yolks.