Monitoring the Anisotropy and Fluidity of the HDL Monolayer as Surrogates of HDL Functionality.

The fluidity of the biological lipid layers modulates processes involved in cardiovascular disease. High-density lipoprotein (HDL) monolayer fluidity is considered as a surrogate of HDL functionality. In particular, the more fluid the HDL monolayer is, the greater the cholesterol efflux (ChE) is observed. Fluidity depends on cholesterol and on the saturation and length of the fatty acids present in lipid layers. Specifically, low cholesterol and short-chain and/or low-saturated fatty acids content in the lipid layers increases fluidity. Lipid peroxidation is also involved in regulating the monolayers' fluidity. HDL oxidation decreases its fluidity and ChE capacity. Accordingly, the presence of antioxidants in biological membranes and in HDL increases fluidity. The fluidity is assessed in polarization studies that measures the steady-state anisotropy (r) using fluorescent probes (such as 1,6-diphenyl-1,3,5-hexatriene; DPH) that mimic the molecular movements of the sample analyzed. Since r refers to the rigidity and fluidity refers to the viscosity of lipid layers, the fluidity index is the inverse value of r (i.e., 1/r). This chapter describes a method for measuring HDL monolayer fluidity and r. The reproducibility of this method was excellent as the intra-assay coefficients of variation (CV) were <2.5 (20 replicates on the same day) and the interassay CV were <5% (60 replicates measured on 3 different days; 20 replicates/day). The method therefore represents a reproducible and useful tool to evaluate HDL functionality as an emerging cardiovascular risk factor.

Variations in daily intakes of myristic and alpha-linolenic acids in sn-2 position modify lipid profile and red blood cell membrane fluidity.

The present study evaluated the effects of moderate intakes of myristic acid (MA), at 1.2% and 1.8% of total energy (TE), associated with a 0.9% TE intake of alpha-linolenic acid (ALA) on lipid and fatty acid profiles and red blood cell membrane fluidity. Twenty-nine monks without dyslipidaemia were enrolled in a 1-year nutritional study in which two experimental diets were tested for 3 months each: diet 1, MA 1.2 % and ALA 0.9%; diet 2, MA 1.8% and ALA 0.9%. A control diet (MA 1.2%, ALA 0.4%) was given 3 months before diets 1 and 2. Thus, two different levels of MA (1.2%, 1.8%) and ALA (0.4%, 0.9%) were tested. Intakes of other fatty acids were at recommended levels. Samples were obtained on completion of all three diets. For fluidity analysis, the red blood cells were labelled with 16-doxylstearate and the probe incorporated the membrane where relaxation-correlation time was calculated. Diet 1 was associated with a decrease in total cholesterol, in LDL-cholesterol, in triacylglycerols and in the ratio of total to HDL-cholesterol; ALA and EPA levels were increased in both phospholipids and cholesterol esters. Diet 2 was associated with a decrease in triacylglycerols and in the ratios of total to HDL-cholesterol and of triacylglycerols to HDL-cholesterol, and with an increase in HDL-cholesterol; EPA levels were decreased in phospholipids and cholesterol esters. Red blood cell membrane fluidity was increased in both diets (P<0.0001), but the higher increase was obtained with diet 1, mainly in the oldest subjects. Intakes of myristic acid (1.2%TE) and ALA (0.9%TE), both mainly in the sn-2 position, were associated with favourable lipid and n-3 long-chain fatty acid profiles. These beneficial effects coexisted with particularly high membrane fluidity, especially among the oldest subjects.

Lipid remodeling of murine epididymosomes and spermatozoa during epididymal maturation.

We have isolated vesicular structures from mouse epididymal fluid, referred to as epididymosomes. Epididymosomes have a roughly spherical aspect and a bilayer membrane, and they are heterogeneous in size and content. They originate from the epididymal epithelium, notably from the caput region, and are emitted in the epididymal lumen by way of apocrine secretion. We characterized their membranous lipid profiles in caput and cauda epididymidal fluid samples and found that epididymosomes were particularly rich in sphingomyelin (SM) and arachidonic acid. The proportion of SM increased markedly during epididymal transit and represented half the total phospholipids in cauda epididymidal epididymosomes. The cholesterol:phospholipid ratio increased from 0.26 in the caput to 0.48 in the cauda epididymidis. Measures of epididymosomal membrane anisotropy revealed that epididymosomes became more rigid during epididymal transit, in agreement with their lipid composition. In addition, we have characterized the membrane lipid pattern of murine epididymal spermatozoa during their maturation. Here, we have shown that mouse epididymal spermatozoa were distinguished by high percentages of SM and polyunsaturated membranous fatty acids (PUFAs), principally represented by arachidonic, docosapentanoic, and docosahexanoic acids. Both SM and PUFA increased throughout the epididymal tract. In particular, we observed a threefold rise in the ratio of docosapentanoic acid. Epididymal spermatozoa had a constant cholesterol:phospholipid ratio (average, 0.30) during epididymal transit. These data suggest that in contrast with epididymosomes, spermatozoal membranes seem to become more fluid during epididymal maturation.

Linoleate supplementation in steers modifies lipid composition of plasma lipoproteins but does not alter their fluidity.

The health value for man of lipids in bovine muscles can be improved by the addition of PUFA to the animals' diets, but such treatments can modify fluidity of plasma lipoproteins and therefore their metabolic functions. The aim of the present study was to analyse whether changes in chemical composition of lipoproteins in steers fed sunflower oil-rich diets altered lipoprotein fluidity, measured by fluorescence polarization and electron spin resonance. LDL, light HDL and heavy HDL fractions were isolated by ultracentrifugation from plasma of eighteen crossbred Charolais x Salers steers. For a period of 70 d, animals were given a control diet (C, n 6) consisting of hay (540 g/kg) and concentrate mixture (460 g/kg) or the same basal diet supplemented with sunflower oil rich in n-6 PUFA (40 g/kg diet DM), given either as crushed seeds (S, n 6) or as a free oil infused directly into the duodenum (O, n 6), thus avoiding ruminal hydrogenation of PUFA. We have shown that in bovine animals: (1) fluidity measurements by fluorescence polarization must be made at the bovine physiological temperature (38.5 degrees C); (2) heavy HDL always appear as the less fluid lipoparticles; (3) electron spin resonance, which does not depend on lipoparticle size, is more appropriate to compare the fluidity of LDL with that of light HDL. The values for lipoprotein fluidity measured by both methods indicated that linoleate-rich diets did not have any effect when compared with diet C; however, chemical variables support a fluidification of lipoparticles, since in steers given the diet O, n-6 PUFA concentrations increased in polar (x1.8) and neutral (x1.6) lipids in lipoparticles (P=0.0001). The phospholipid:protein ratio increased in light (+20 %, P=0.019) and heavy (+23 %, P=0.06) HDL and especially in LDL (+46 %, P=0.0001); the total cholesterol:phospholipid ratio decreased in the three lipoprotein classes (-15 to -30 %, NS). Diet S led to similar but less pronounced effects. We concluded that linoleate-rich diets modified the chemical composition of plasma lipoproteins in steers, but did not alter their fluidity; this probably occurred as a result of 'homeoviscous adaptation', which ensured their functional capacity.

Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization.

Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages.

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.

Treatment of the rat hepatic stellate cell line, PAV-1, by retinol and palmitic acid leads to a convenient model to study retinoids metabolism.

The main site of vitamin A storage in the liver is the hepatic stellate cells (HSC). Involvement of HSC in vitamin A metabolism has mainly been studied using primary culture, which represents the most physiological model but technically suffers several drawbacks (yield, low reproducibility, etc.). To circumvent these problems, we have previously established and characterised an immortalised rat HSC line named PAV-1. This study aimed to investigate in PAV-1 and in primary HSC (i) the incorporation of retinol and its esterification, (ii) the cellular retinol-binding protein (CRBP) content, (iii) the acid retinyl ester hydrolase activity (aREH), (iv) the thermal susceptibility and (v) the lipid composition of the membranes, which may play a crucial role in retinol transport across cellular membrane. In routine conditions of culture, the rate of retinol esterification in PAV-1 was low (5.2%) compared to that obtained with primary HSC (69.9%). Retinol pre-treatment doubled this esterification rate (10.7%) and the CRBP content in PAV-1. The co-incubation with retinol and palmitic acid enabled PAV-1 to esterify retinol with a rate close to that of primary HSC (66.2% vs. 69.9%) and with similar retinyl ester profiles. aREH activity was higher in primary HSC than in PAV-1. Thermal susceptibility and phospholipid composition of membranes in PAV-1 treated cells were similar to those of primary HSC. In conclusion, our study shows that PAV-1 cells treated with retinol and palmitic acid is a sound and convenient model for studying vitamin A mobilisation, a fundamental physiological event occurring in HSC.