Hepatoprotective effects of sea cucumber ether-phospholipids against alcohol-induced lipid metabolic dysregulation and oxidative stress in mice.

Sea cucumber is widely consumed as food and folk medicine in Asia, and its phospholipids are rich sources of dietary eicosapentaenoic acid enriched ether-phospholipids (ether-PLs). Emerging evidence suggests that ether-PLs are associated with neurodegenerative disease and steatohepatitis. However, the function and mechanism of ether-PLs in alcoholic liver disease (ALD) are not well understood. To this end, the present study sought to investigate the hepatoprotective effects of sea cucumber ether-PLs, including plasmenyl phosphatidylethanolamine (PlsEtn) and plasmanyl phosphatidylcholine (PlsCho), and their underlying mechanisms. Our results showed that compared with EtOH-induced mice, ether-PL treated mice showed improved liver histology, decreased serum ALT and AST levels, and reduced alcohol metabolic enzyme (ALDH2 and ADH1) expressions. Mechanistic studies showed that ether-PLs attenuated "first-hit" hepatic steatosis and lipid accumulation evoked by alcohol administration. Moreover, PlsEtn more effectively restored endogenous plasmalogen levels than PlsCho, thereby enhancing hepatic antioxidation against "second-hit" reactive oxygen species (ROS) due to the damaged mitochondria and abnormal ethanol metabolism. Taken together, sea cucumber ether-PLs show great potential to become a natural functional food against chronic alcohol-induced hepatic steatosis and lipid metabolic dysregulation.

Enzymatic activity of Lecithin:retinol acyltransferase: a thermostable and highly active enzyme with a likely mode of interfacial activation.

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70°C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated ß-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100°C. The results of this study highly improved our understanding of this enzyme.

Structural elucidation of molecular species of pacific oyster ether amino phospholipids by normal-phase liquid chromatography/negative-ion electrospray ionization and quadrupole/multiple-stage linear ion-trap mass spectrometry.

Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.

Impedance analysis of valinomycin activity in nano-BLMs.

Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.

Release of free F2-isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet-activating factor acetylhydrolases.

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Ultraviolet B radiation generates platelet-activating factor-like phospholipids underlying cutaneous damage.

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.

Reinvestigation by phosphorus NMR of lipid distribution in bicelles.

Mixtures of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC) in water form disks also called bicelles and different bilayer organizations when the mol ratio of the two lipids and the temperature are varied. The spontaneous alignment in a magnetic field of these bilayers above the transition temperature T(m) of DMPC is an attractive property that was successfully used to investigate protein structure by NMR. In this article, we have attempted to give an overview of all structural transformations of DMPC/DHPC mixtures that can be inferred from broad band (31)P-NMR spectroscopy between 5 and 60 degrees C. We show that above a critical temperature, T(v), perforated vesicles progressively replace alignable structures. The holes in these vesicles disappear above a new temperature threshold, T(h). The driving force for these temperature-dependent transformations that has been overlooked in previous studies is the increase of DHPC miscibility in the bilayer domain above T(m). Accordingly, we propose a new model (the "mixed bicelle" model) that emphasizes the consequence of the mixing. This investigation shows that the various structures of DMPC in the presence of increasing mol ratios of the short-chain DHPC is reminiscent of the observation put forward by several laboratories investigating solubilization and reconstitution of biological membranes.

Solid-state NMR spectroscopic studies of an integral membrane protein inserted into aligned phospholipid bilayer nanotube arrays.

This communication demonstrates for the first time that solid-state NMR spectroscopic studies can be used to investigate aligned phospholipid bilayer nanotube arrays. Also, an integral membrane peptide can be successfully incorporated into the oriented phospholipid bilayer nanotube arrays and studied with 2H solid-state NMR spectroscopy.

Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo[a]pyrene to the activated o-quinone metabolite benzo[a]pyrene-7,8-dione.

The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols. Previous studies demonstrated that rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD/DD), a member of the aldo-keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling o-quinones. Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP+ -dependent oxidation of PAH trans-dihydrodiols. In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR. The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized. Calculation of KM and kcat values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3alpha-HSD (DDX) proteins. The ability of human DDs to oxidize the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7, 8-dihydrobenzo[a]pyrene (BP-diol) was then examined. A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (+/-)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX. Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S- and major (-)-R,R-stereoisomers formed in vivo are substrates. First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S, 8S-enantiomer. The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo[a]pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ. Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.

Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent.

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH3 could be stably associated with liposomes, less free ET-18-OCH3 would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH3. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH3 liposome compositions were minimally hemolytic. For example, whereas the HI5 (drug concentration required to cause 5% human red cell lysis) was 5-6 microM for free ET-18-OCH3, it was approximately 250 microM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH3, (4:3:1:2) and 640 microM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH3 (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH3 in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH3.

Combination of antitumor ether lipid with lipids of complementary molecular shape reduces its hemolytic activity.

Because the therapeutic use of the antitumor ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) is restricted by its hemolytic activity we explored the use of lipid packing parameters to reduce this toxicity by creating structurally optimized ET-18-OCH3 liposomes. We postulated that combination of ET-18-OCH3, which is similar in structure to lysophosphatidylcholine, with lipid molecules of complementary molecular shape (opposite headgroup/chain volume) would likely yield a stable lamellar phase from which ET-18-OCH3 exchange to red blood cell membranes would be curtailed. To quantitate the degree of shape complementarity, we used a Langmuir trough and measured the mean molecular area per molecule (MMAM) for monolayers comprised of ET-18-OCH3, the host lipids, and binary mixtures of varying mole percentage ET-18-OCH3. The degree of complementarity was taken as the reduction in MMAM from the value expected based on simple additivity of the individual components. The greatest degree of shape complementarity was observed with cholesterol: the order of complementarity for the ET-18-OCH3-lipid mixtures examined was cholesterol >> DOPE > POPC approximately DOPC. Phosphorus NMR and TLC analysis of aqueous suspensions of ET-18-OCH3 (40 mol%) with the host lipids revealed them to all be lamellar phase. For ET-18-OCH3 at 40 mol% in liposomes, the hemolytic activity followed the trend of the reduction in MMAM and was least for the ET-18-OCH3/cholesterol system (H50 = 661 microM ET-18-OCH3) followed by ET-18-OCH3/DOPE (H50 = 91 microM) and mixtures with POPC and DOPC which were comparable at H50 = 26 microM and 38 microM, respectively: the H50 concentration for free ET-18-OCH3 was 16 microM. This experimental strategy for designing optimized liposomes with a reduction in exchange, and hence toxicity, may be useful for other amphipathic/lipophilic drugs that are dimensionally compatible with lipid bilayers.

Solid state 13C NMR of unlabeled phosphatidylcholine bilayers: spectral assignments and measurement of carbon-phosphorus dipolar couplings and 13C chemical shift anisotropies.

The direct measurement of 13C chemical shift anisotropies (CSA) and 31P-13C dipolar splitting in random dispersions of unlabeled L alpha-phase phosphatidylcholine (PC) has traditionally been difficult because of extreme spectral boradening due to anisotropy. In this study, mixtures of dimyristoyl phosphatidylcholine (DMPC) with three different detergents known to promote the magnetic orientation of DMPC were employed to eliminate the powder-pattern nature of signals without totally averaging out spectral anisotropy. The detergents utilized were CHAPSO, Triton X-100, and dihexanoylphosphatidylcholine (DHPC). Using such mixtures, many of the individual 13C resonances from DMPC were resolved and a number of 13C-31P dipolar couplings were evident. In addition, differing line widths were observed for the components of some dipolar doublets, suggestive of dipolar/chemical shift anisotropy (CSA) relaxation interference effects. Oriented sample resonance assignments were made by varying the CHAPSO or DHPC to DMPC ratio to systematically scale overall bilayer order towards the isotropic limit. In this manner, peaks could be identified based upon extrapolation to their isotropic positions, for which assignments have previously been made (Lee, C.W.B., and R.G. Griffin. 1989. Biophys. J. 55:355-358; Forbes, J., J. Bowers, X. Shan, L. Moran, E. Oldfield, and M.A. Moscarello. 1988. J. Chem. Soc., Faraday, Trans. 1 84:3821-3849). It was observed that the plots of CSA or dipolar coupling versus overall bilayer order obtained from DHPC and CHAPSO titrations were linear. Estimates of the intrinsic dipolar couplings and chemical shift anisotropies for pure DMPC bilayers were made by extrapolating shifts and couplings from the detergent titrations to zero detergent. Both detergent titrations led to similar "intrinsic" CSAs and dipolar couplings. Results extracted from an oriented Triton-DMPC mixture also led to similar estimates for the detergent-free DMPC shifts and couplings. The results from these experiments were found to compare favorably with limited measurements made from pure L alpha PC. This detergent-based method for assigning spectra and for determining dipolar couplings and CSA in detergent-free systems should be extendable to other lipid systems. The resulting data set from this study may prove useful in future modeling of the structure and dynamics of DMPC bilayers. In addition, the fact that experiments utilizing each of the three detergents led to similar estimates for the spectral parameters of pure DMPC, and the fact that spectral parameter versus bilayer order plots were linear, indicate that the averaged conformation and dynamics of DMPC in the presence of the three detergents are very similar to those of pure L alpha DMPC.

Similarities in surface lipids of chylomicrons from glyceryl and alkyl ester feeding: major components.

This study tests the hypothesis that the rat chylomicrons are assembled and released into lymph similarly regardless of the site (rough or smooth endoplasmic reticulum) or pathway (phosphatidic acid or monoacylglycerol) of triacylglycerol biosynthesis. For this purpose we determined the lipid class, fatty acid and molecular species composition of the choline, ethanolamine, inositol and serine phospholipids of lymph chylomicrons during absorption of menhaden, mustard-seed and corn oil (monoacylglycerol pathway) or the corresponding fatty acid methyl or ethyl esters (phosphatidic acid pathway). The dietary fatty acids were found to be incorporated to various extents into different phospholipid classes, the proportions of which were not affected by the nature of the dietary fat. The chylomicron phospholipids contained 80-82% choline, 8% ethanolamine and 2.5% inositol glycerophospholipids, and much smaller amounts of serine and other minor phospholipids. Administration of a meal of each dietary fat resulted in a retention of approximately 50% endogenous fatty acids in the major glycerophospholipids of the chylomicrons. A minimum of 50% of the molecular species of the choline and ethanolamine glycerophospholipids contained at least one exogenous fatty acid. No significant discrepancies were found in the fatty acid and molecular species composition of the glycerophospholipids between chylomicrons from the oil and corresponding ester feeding. It is concluded that the chylomicrons arising from the monoacylglycerol (oil feeding) and the phosphatidic acid (ester feeding) pathways of triacylglycerol biosynthesis become enveloped in surfactant monolayers containing qualitatively and quantitatively identical classes and molecular species of phospholipids.