Martine Couturier

Preferential Sphingosine-1-Phosphate Enrichment and Sphingomyelin Depletion Are Key Features of Small Dense HDL3 Particles. Relevance to Antiapoptotic and Antioxidative Activities

Objective—The purpose of this study was to define heterogeneity in the molecular profile of lipids, including sphingomyelin and sphingosine-1-phosphate, among physicochemically-defined HDL subpopulations and potential relevance to antiatherogenic biological activities of dense HDL3. Methods and Results—The molecular profile of lipids (cholesteryl esters, phospholipids, sphingomyelin, and sphingosine-1-phosphate) in physicochemically-defined normolipidemic HDL subpopulations was determined by high-performance liquid chromatography and gas chromatography. As HDL particle size and molecular weight decreased with increment in density, molar lipid content diminished concomitantly. On a % basis, sphingomyelin abundance diminished in parallel with progressive increase in HDL density from HDL2b (12.8%) to HDL3c (6.2%; P<0.001); in contrast, sphingosine-1-phosphate was preferentially enriched in small HDL3 (40 to 50 mmol/mol HDL) versus large HDL2 (15 to 20 mmol/mol HDL; P<0.01). Small HDL3c was equally enriched in LpA-I particles relative to LpA-I:A-II. The sphingosine-1-phosphate/sphingomyelin ratio correlated positively with the capacities of HDL subspecies to attenuate apoptosis in endothelial cells (r=0.73, P<0.001) and to retard LDL oxidation (r=0.58, P<0.01). Conclusions—An elevated sphingosine-1-phosphate/sphingomyelin ratio is an integral feature of small dense HDL3, reflecting enrichment in sphingosine-1-phosphate, a key antiapoptotic molecule, and depletion of sphingomyelin, a structural lipid with negative impact on surface fluidity and LCAT activity. These findings further distinguish the structure and antiatherogenic activities of small, dense HDL.

HDL3-Mediated Inactivation of LDL-Associated Phospholipid Hydroperoxides Is Determined by the Redox Status of Apolipoprotein A-I and HDL Particle Surface Lipid Rigidity: Relevance to Inflammation and Atherogenesis

Objectives—Small dense HDL3 particles of defined lipidome and proteome potently protect atherogenic LDL against free radical–induced oxidation; the molecular determinants of such antioxidative activity in these atheroprotective, antiinflammatory particles remain indeterminate. Methods and Results—Formation of redox-active phosphatidylcholine hydroperoxides (PCOOH) and redox-inactive phosphatidylcholine hydroxides (PCOH) was initiated in LDL by free radical–induced oxidation. Human HDL3 inactivated LDL-derived PCOOH (−62%, P<0.01) and enhanced accumulation of PCOH (2.1-fold, P<0.05); in parallel, HDL3 accumulated minor amounts of PCOOH. Enzyme-deficient reconstituted dense HDL potently inactivated PCOOH (−43%, P<0.01). HDL3-mediated reduction of PCOOH to PCOH occurred concomitantly with oxidation of methionine residues in HDL3-apolipoprotein AI (apoAI). Preoxidation of methionine residues by chloramine T markedly attenuated PCOOH inactivation (−35%); by contrast, inhibition of HDL3-associated enzymes was without effect. PCOOH transfer rates from oxidized LDL to phospholipid liposomes progressively decreased with increment in the rigidity of the phospholipid monolayer. Conclusions—The redox status of apoAI and surface lipid rigidity represent major determinants of the potent HDL3-mediated protection of LDL against free radical–induced oxidation. Initial transfer of PCOOH to HDL3 is modulated by the surface rigidity of HDL3 particles with subsequent reduction of PCOOH to PCOH by methionine residues of apoAI.

Small, dense HDL 3 particles attenuate apoptosis in endothelial cells: pivotal role of apolipoprotein A-I

Plasma high‐density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low‐density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC‐1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC‐1 against oxLDL‐induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase‐3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis‐inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti‐oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A‐I (apoA‐I) predominated, accounted for ∼70% of HDL anti‐apoptotic activity. Furthermore, HDL reconstituted with apoA‐I, cholesterol and phospholipid potently protected HMEC‐1 from apoptosis. By contrast, modification of the content of sphingosine‐1‐phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid‐poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA‐I is pivotal to such protection.

Cholesteryl Ester Hydroperoxide Lability Is a Key Feature of the Oxidative Susceptibility of Small, Dense LDL

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

Evidence of the Formation of Different Hydroperoxides in Irradiated Gamma-Linolenate Solutions: Effect of Micelle Formation

Abstract Hindo, J., Hauville, C., Rémita, S., Thérond, P., Couturier, M., Jore, D. and Gardès-Albert, M. Evidence of the Formation of Different Hydroperoxides in Irradiated Gamma-Linolenate Solutions: Effect of Micelle Formation. Radiat. Res. 153, 201–207 (2000). Peroxidation of unconjugated polyunsaturated fatty acids such as linolenic acid proceeds through a free radical chain mechanism and is accompanied by the formation of conjugated dienes such as hydroperoxides. In an investigation of radiation-induced oxidation of aqueous linolenate, we have measured two indexes of peroxidation: (1) conjugated dienes by means of absorption spectroscopy and (2) hydroperoxides by high-pressure liquid chromatography using detection of chemiluminescence. The experimental results indicate a strong effect of the concentration of linolenate on the yields of oxidized products. In addition, this work shows the quantitative production of two kinds of hydroperoxides. The ratio of these hydroperoxides is independent of the radiation dose but is dependent on the linolenate concentration. One hydroperoxide is formed predominantly below the critical micellar concentration (3 mM under our conditions), while the second is observed predominantly when micelles are formed in the aqueous medium. The influence of the composition of the medium on the nature of both hydroperoxides is discussed.

Determination of the Yield of Radiation-Induced Peroxidation of Sodium Linoleate in Aqueous Monomeric and Micellar Solutions

Peroxidation of polyunsaturated fatty acids such as linoleic acid in aqueous micellar solution proceeds through a free-radical chain mechanism and is accompanied by the formation of conjugated dienes, some in the form of hydroperoxides. In the course of an investigation of radiation-induced oxidation of aqueous sodium linoleate, we have measured three indexes of peroxidation-conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances-by means of absorption spectroscopy, high-pressure liquid chromatography and spectrofluorimetry, respectively. There are linear correlations between the amounts of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances. The radiolytic yields have been determined from the radiation dose dependence of the three markers of peroxidation as a function of sodium linoleate concentration. The results obtained indicate a strong effect of the concentrations of oxygen and linoleate on the yields of the products. The yields at different lipid concentrations display a large increase in chain propagation length; this is discussed in terms of the effect of micellar size.

Small, dense high-density lipoprotein 3 particles exhibit defective antioxidative and anti-inflammatory function in familial hypercholesterolemia: Partial correction by low-density lipoprotein apheresis

BACKGROUND Familial hypercholesterolemia (FH) features elevated oxidative stress and accelerated atherosclerosis driven by elevated levels of atherogenic lipoproteins relative to subnormal levels of atheroprotective high-density lipoprotein (HDL). Small, dense HDL3 potently protects low-density lipoprotein (LDL) against proinflammatory oxidative damage. OBJECTIVE To determine whether antioxidative and/or anti-inflammatory activities of HDL are defective in FH and whether such defects are corrected by LDL apheresis. METHODS Antioxidative and antiinflammatory activities of HDL were evaluated as protection of reference LDL from oxidative stress and capacity to prevent accumulation of proinflammatory oxidised lipids, respectively. Lipid surface rigidity of HDL was assessed using a fluorescent probe. HDL components were measured by analytical approaches. Systemic oxidative stress was characterized as plasma 8-isoprostanes. RESULTS Pre-LDL-apheresis, FH patients (n = 10) exhibited elevated systemic oxidative stress (3.3-fold, P < 0.001) vs. sex- and age-matched normolipidemic controls (n = 10). Both antioxidative and antiinflammatory activity of HDL3 were impaired (up to -91%, P < 0.01) in FH. Sphingomyelin and saturated fatty acid contents were elevated in FH HDL3, resulting in enhanced lipid surface rigidity. The surface lipid content (phospholipids, free cholesterol) was reduced in FH (up to -15%, P < 0.001), whereas content of core lipids (cholesteryl esters, triglycerides) was elevated (up to +17%, P < 0.001). Molar apolipoprotein A-I content of HDL3 was subnormal in FH. A single LDL-apheresis session partially corrected (by up to 76%) deficient HDL antiatherogenic activities, attenuated systemic oxidative stress and partially normalised both the lipid composition and surface rigidity of HDL particles. CONCLUSIONS FH features elevated oxidative stress and deficient antioxidative and anti-inflammatory activities of small, dense HDL3; such functional deficiency is intimately linked to anomalies in lipid and protein composition, which may impair the capacity of HDL to acquire and inactivate oxidized lipids.

Standard Treatment of α-Tocopherol in Alagille Patients with Severe Cholestasis Is Insufficient

Alpha-tocopherol (α-T) is the most effective lipid-soluble antioxidant present in cells. We investigated the efficacy of α-T supplements for preventing lipid peroxidation in patients with Alagille syndrome, according to the severity of cholestasis. Patients were assigned to two groups on the basis of plasma bilirubin concentration (group I, bilirubin <100 μM; group II, bilirubin >100 μM). α-T concentrations were determined in plasma, in isolated lipoproteins, and in red blood cell membranes. In both groups of patients, α-T concentrations in plasma were similar to those in control subjects, but the distribution of α-T in lipoproteins was affected by the abnormal lipoprotein pattern in these patients. The efficacy of α-T was estimated by determining the amount of hydroperoxide produced from phosphatidylcholine and phosphatidylethanolamine (PE) molecular species owing to oxidative stress induced by lipoxygenase treatment. The concentrations of phosphatidylcholine molecular species and its corresponding hydroperoxides were significantly higher in both groups of patients. In group I, α-T and PE molecular species concentrations were similar to those in control subjects, but PE hydroperoxide concentrations were higher than those in the control subjects. In group II, α-T concentration was significantly lower and the concentrations of some PE molecular species and all PE hydroperoxides were lower than those in the control subjects. In conclusion, erythrocyte membrane α-T concentration was significantly lower only in patients with severe jaundice, despite α-T supplementation, raising the question as to whether the usual treatment was appropriate in this group.

Radiolytic yield of cardiolipin peroxidation by gamma rays in large unilamellar vesicles of phosphatidylcholine.

Abstract Sid Ahmed-Adrar, N., Collin, F., Couturier, M., Vitrac, H., Bonnefont-Rousselot, D., Jore, D. and Gardès-Albert, M. Radiolytic Yield of Cardiolipin Peroxidation by Gamma Rays in Large Unilamellar Vesicles of Phosphatidylcholine. Radiat. Res. 171, 622–630 (2009). Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1′,3′-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min−1). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 ± 0.2) × 10−7 mol J−1 for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(–CL) = (2.8 ± 0.1) × 10−7 mol J−1] and the formation of CL hydroperoxides [G(CLOOHT) = (3.1 ± 0.2) × 10−7 mol J−1]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 ± 0.1) × 10−7 mol J−1 and (2.8 ± 0.1) × 10−7 mol J−1, respectively], their sum [(4.3 ± 0.2) × 10−7 mol J−1] being higher than GHO· (2.8 × 10−7 mol J−1). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOHT) ∼ 0] and the yield of disappearance of PLPC [(1.5 ± 0.1) × 10−7 mol J−1], likely because lipid fragments (not measured in this work) could be generated from HO· reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10–15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO·-induced peroxidation of PLPC.

Human sperm quality and lipid content after migration into normal ovulatory human cervical mucus containing low numbers of leukocytes.

The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containing>or=10(4) leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation.