Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

A new 5α,8α-epidioxysterol with immunosuppressive activity from the South China Sea soft coral Sinularia sp.

A new 5α,8α-epidioxysterol, namely yalongsterol A (1), along with two known related steroids 5α,8α-epidioxy-24-methyl-cholesta-6,24(28)-dien-3ß-ol (2) and (22E,24S)-5α,8α-epidioxy-24-methyl-cholesta-6,22-dien-3ß-ol (3), were isolated from the South China Sea soft coral Sinularia sp. Their structures were established by extensive spectroscopic analyses and comparisons of the spectral data with those reported in the literature. In bioassay, compounds 1-3 showed moderate immunosuppressive activities against T and/or B lymphocyte cells with IC50 values ranging from 19.30 to 59.49 µM, and low cytotoxicity on murine splenocytes with CC50 values ranging from 40.88 to 62.29 µM.[Formula: see text].

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Direct Measurement of Free and Esterified Cholesterol Mass in Differentiated Human Podocytes: A TLC and Enzymatic Assay-Based Method.

Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.

Entamoeba mitosomes play an important role in encystation by association with cholesteryl sulfate synthesis.

Hydrogenosomes and mitosomes are mitochondrion-related organelles (MROs) that have highly reduced and divergent functions in anaerobic/microaerophilic eukaryotes. Entamoeba histolytica, a microaerophilic, parasitic amoebozoan species, which causes intestinal and extraintestinal amoebiasis in humans, possesses mitosomes, the existence and biological functions of which have been a longstanding enigma in the evolution of mitochondria. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. However, because the final metabolites of sulfate activation remain unknown, the overall scheme of this metabolism and the role of mitosomes in Entamoeba have not been elucidated. In this study we purified and identified cholesteryl sulfate (CS) as a final metabolite of sulfate activation. We then identified the gene encoding the cholesteryl sulfotransferase responsible for synthesizing CS. Addition of CS to culture media increased the number of cysts, the dormant form that differentiates from proliferative trophozoites. Conversely, chlorate, a selective inhibitor of the first enzyme in the sulfate-activation pathway, inhibited cyst formation in a dose-dependent manner. These results indicate that CS plays an important role in differentiation, an essential process for the transmission of Entamoeba between hosts. Furthermore, we show that Mastigamoeba balamuthi, an anaerobic, free-living amoebozoan species, which is a close relative of E. histolytica, also has the sulfate-activation pathway in MROs but does not possess the capacity for CS production. Hence, we propose that a unique function of MROs in Entamoeba contributes to its adaptation to its parasitic life cycle.

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase.

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

Occurrence of cytotoxic 9-oxononanoyl secosterol aldehydes in human low-density lipoprotein.

The reaction products of three major cholesteryl esters, cholesteryl palmitate (C16:0-CE), cholesteryl oleate (C18:1-CE), and cholesteryl linoleate (C18:2-CE), present in human low-density lipoprotein (LDL) treated with ozone were isolated and characterized. In vitro ozonization of C16:0-CE was found to form the palmitoyl ester of secosterol-A (3ß-hydroxy-5-oxo-5,6-secocholestan-6-al) and its aldolization product secosterol-B (3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde). On the other hand, when C18:1-CE and C18:2-CE were oxidized by ozone, the aldehyde 9-oxononanoyl cholesterol (9-ONC) was formed as a primary product, which was then further oxidized to form 9-oxononanoyl secosterol-A (9-ON-secoA) and 9-oxononanoyl secosterol-B (9-ON-secoB). The compounds 9-ON-secoA and -B, but not 9-ONC, were found to exhibit strong cytotoxicity against human leukemia HL-60 cells. An LC-ESI-MS/MS method was developed for the detection of these cholesteryl ester ozonolysis products by derivatizing them with dansyl hydrazine. Using this method, we found for the first time that low concentrations of 9-ON-secoA and -B, but not palmitoyl secosterols, were present in human LDL. These novel oxidized cholesterol esters, 9-ON-secoA and -B, probably play important roles in the pathogenesis of several inflammatory disorders such as cancer, diabetes, atherosclerosis, and neurodegenerative diseases.

Evolving neural network optimization of cholesteryl ester separation by reversed-phase HPLC.

Cholesteryl esters have antimicrobial activity and likely contribute to the innate immunity system. Improved separation techniques are needed to characterize these compounds. In this study, optimization of the reversed-phase high-performance liquid chromatography separation of six analyte standards (four cholesteryl esters plus cholesterol and tri-palmitin) was accomplished by modeling with an artificial neural network-genetic algorithm (ANN-GA) approach. A fractional factorial design was employed to examine the significance of four experimental factors: organic component in the mobile phase (ethanol and methanol), column temperature, and flow rate. Three separation parameters were then merged into geometric means using Derringer's desirability function and used as input sources for model training and testing. The use of genetic operators proved valuable for the determination of an effective neural network structure. Implementation of the optimized method resulted in complete separation of all six analytes, including the resolution of two previously co-eluting peaks. Model validation was performed with experimental responses in good agreement with model-predicted responses. Improved separation was also realized in a complex biological fluid, human milk. Thus, the first known use of ANN-GA modeling for improving the chromatographic separation of cholesteryl esters in biological fluids is presented and will likely prove valuable for future investigators involved in studying complex biological samples.

Antibacterial triterpenes from the threatened wood-decay fungus Fomitopsis rosea.

From a biologically active extract from the fungus Fomitopsis rosea, two new lanostane triterpenes were isolated, 3alpha-(3'-butylcarboxyacetoxy)oxepanoquercinic acid C 1 and 3alpha-hydroxy-24-methylene-23-oxolanost-8-en-26-carboxylic acid 2, along with three known triterpenes, 3-5, and the epidioxy sterol derivative 6. The structures of the compounds were elucidated based on their spectral properties. All triterpenes demonstrated antibacterial activity against S. aureus but possessed no antiradical activity against DPPH radicals.

5alpha,8alpha-Epidioxysterols from the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum: isolation and evaluation of their antiproliferative activity.

Three new (1, 4, 9) and nine previously reported (2, 3, 5-8, 10-12) 5alpha,8alpha-epidioxysterols were isolated from the organic extracts of the gorgonian Eunicella cavolini and the ascidian Trididemnum inarmatum. The structures and relative configurations of 1-12 were established on the basis of detailed NMR spectroscopic analyses and comparison with the literature. The growth inhibitory effects of 1-12 were evaluated against MCF-7 human breast cancer cells. Compound 1, bearing a cyclopropyl moiety in the side chain, exhibited the highest antiproliferative activity.

Properties of the products formed by the activity of serum opacity factor against human plasma high-density lipoproteins.

Serum opacity factor from Streptococcus pyogenes transfers the cholesteryl esters (CE) of approximately 100,000 plasma high-density lipoprotein particles (HDL) to a CE-rich microemulsion (CERM) while forming neo HDL, a cholesterol-poor HDL-like particle. HDL, neo HDL, and CERM are distinct. Neo HDL is lower in free cholesterol and has lower surface and total microviscosities than HDL; the surface polarity of neo HDL and HDL are similar. CERM is much larger than HDL and richer in cholesterol and CE. Although the surface microviscosity of HDL is higher than that of CERM, they have similar total microviscosities because cholesterol partitions into the neutral lipid core. Because of its unique surface properties apo E preferentially associates with the CERM. In contrast, the composition and properties of neo HDL make it a potential acceptor of cellular cholesterol and its esterification. Thus, neo HDL and CERM are possible vehicles for improving cholesterol transport to the liver.

Spectral editing of organic mixtures into pure components using NMR spectroscopy and ultraviscous solvents.

A general technique is described that permits the extraction of a complete 1H NMR spectrum for components in organosoluble mixtures. The approach should find a wide range of applications considering that pure component spectra can be generated without the need for physical separation. This technique is especially significant for synthetic organic chemistry and the pharmaceutical industry due to the potential to isolate a product spectrum even in the presence of overlapping starting materials, byproducts, or degradation products. A viscous oil-based solvent system that can be temperature-manipulated from essentially a solid at one extreme to a freely flowing liquid at the other is employed. The system contains no protons and is miscible with common organic solvents. Through careful control of the temperature and thus solvent viscosity, the behavior of small molecules moves from the positive to the extreme of the negative NOE regime. Under such conditions, all protons in a molecule correlate with all other protons as propagation by spin diffusion becomes highly efficient, behavior normally only observed with rigid macromolecules in conventional solvents. Therefore, as long as one proton (or carbon signal in hybrid experiments) is resolved for a component in a mixture, the entire proton spectrum for that molecule can be cleanly extracted from a 2D NOESY spectrum (or from selective 1D NOE-based analogues). Preliminary results are highly encouraging, indicating that the approach may be feasible for a wide range of molecules and mixtures; however, in practice the exact types of structures, combinations of structures, and range of concentrations that can be cleanly extracted will become evident as the technique becomes better established.

[Studies on chemical constituents of marine bryozoan Bugula neritina L].

Seven compounds were isolated from the marine bryozoan Bugula neritina L. Their chemical structures were identified by NMR and MS spectroscopies as follows: cholesterol (I), cholest-4-en-3-one (II), cholesteryl myristate (III), 3beta,5alpha,9alpha-trihydrox-y-(22E,24R)-ergosta-,22-dien-6-one (IV), 3beta,5alpha,6beta-trihydroxy-(22E,24R)-ergosta-7,22-dien (V), uracil (VI), thymine (VII). Compounds II-VII were isolated from Bugula neritina L. for the first time.

New biologically active epidioxysterols from Stereum hirsutum.

From the fungus Stereum hirsutum have been isolated and identified two new epidioxysterols 1, 4, together with two known ones 2 and 3. Their structures were elucidated on the basis of spectroscopic analysis and chemical reactions. Epidioxysterols 1-4 have been shown to possess a significant activity against Mycobacterium tuberculosis.

A new cytotoxic cholesterol sulfate from marine sponge Halichondria rugosa.

A new steroid, 24xi,25-dimethyl-3alpha-hydroxyl-cholest-5-ene-2beta-ol sodium sulfate (1), together with a known steroid, 24xi,25-dimethyl-cholest-5-ene-2beta,3alpha-diol disodium sulfate (2), was isolated from the ethanol extract of marine sponge Halichondria rugosa. Their structures were elucidated on the base of spectroscopic analysis. Both compounds showed cytotoxicity to four human cancer cell lines (BEL-7402, HT-29, SPC-A1 and U-251) with IC(50) values between 6.5 and 23.1 microM.

Lipoprotein-like particles and cholesteryl esters in human Bruch's membrane: initial characterization.

PURPOSE: To isolate and characterize cholesteryl ester-containing, lipoprotein-like particles (LLPs) from normal aged human Bruch's membrane (BrM)/choroid (Ch). METHODS: From BrM/Ch of 20 eyes of 10 donors aged >60 years, LLPs were released by high-salt buffer, fractionated by density gradient ultracentrifugation, and characterized by determining cholesterol, triglyceride, and phospholipid concentration (by enzymatic colorimetry and fluorometry); cholesteryl ester composition (by electrospray ionization mass spectrometry, ESI/MS); and particle morphology (by negative stain electron microscopy). Apolipoprotein (apo) gene expression was determined with RT-PCR, Western blot analysis, and immunofluorescence of retinal-choroidal cryosections. In paraformaldehyde-preserved eyes (20 eyes of 20 donors), cholesteryl ester composition of BrM/Ch, cornea, and sclera was determined by ESI/MS. RESULTS: A pooled fraction of LLP released from BrM/Ch (concentrated total LLP, density [d] < 1.24 g/mL fraction) was fractionated into two peaks. A large Peak 1 (with plasma LDL-HDL density range), containing predominantly phospholipid and unesterified cholesterol, was morphologically heterogeneous. A small Peak 2 (with plasma VLDL density range), enriched with esterified cholesterol, contained approximately 100 nm diameter round electron-lucent particles. Both peaks contained apoB and apoA-I, RPE and retina contained apoA-I mRNA transcripts, and BrM and drusen contained apoA-I immunoreactivity. Peaks 1 and 2, native RPE, and fresh BrM/Ch were cholesteryl linoleate enriched and contained little cholesteryl docosahexaenoate. Preserved BrM/Ch was cholesteryl oleate-enriched, unlike sclera and cornea. CONCLUSIONS: BrM/Ch LLP do not resemble plasma lipoproteins in density profile, cholesterol distribution, or morphology. Peak 2 contains EC-rich LLP resembling BrM particles in situ. BrM/Ch cholesteryl esters respond to long-term storage differently than esters of plasma lipoprotein origin accumulated in other ocular tissues. Evidence of intraocular apoB and apoA-I expression supports an emerging hypothesis that the RPE assembles and secretes a large, possibly novel, lipoprotein particle.

Wolman disease and cholesteryl ester storage disease diagnosed by histological and ultrastructural examination of intestinal and liver biopsy.

Deficient activity of lysosomal acid lipase (LAL) results in massive accumulation of cholesteryl esters and triglycerides in most tissues of the body. The deficiency state is expressed in two major phenotypes: Wolman disease (WD) and cholesteryl ester storage disease (CESD). WD occurs in infancy and is nearly always fatal before the age of 1 year, whereas CESD can be more benign and may not be detected until adulthood. Since there are no specific routine laboratory observations that suggest these metabolic diseases, diagnosis is based on the clinical picture combined with LAL deficiency in cultured skin fibroblasts or peripheral lymphocytes. Both disorders are rather rare, considering that about a hundred of cases have been described up to now. This study describes the histological and ultrastructural aspects disclosed by intestinal or liver biopsy in three cases of WD and in two cases of CESD. Furthermore, it emphasizes the role of morphological findings in pointing the diagnosis towards a metabolic storage disease.

On-column electrochemical reactions accompanying the electrospray process.

It is well-established that electrochemical reactions can occur within the capillary of an electrospray (ES) devise coupled to a mass spectrometer. In fact, such reactions must occur to maintain charge balance during the ES process. However, electrochemical reactions occurring distal to the capillary as a result of the high potential applied to the capillary have not been thoroughly investigated. In the present communication, we show that electrochemical processes can occur on a high performance liquid chromatography (HPLC) column coupled to an ES capillary. On-column solvent electrolysis is proposed to generate free radicals, which can subsequently initiate analyte oxidation. Oxidation of steroid sulfates possessing a reactive double bond between C-5 and C-6 is demonstrated. The possibility of similar reactions occurring within peptides possessing a site of unsaturation is also considered.

Beta-glucan fractions from barley and oats are similarly antiatherogenic in hypercholesterolemic Syrian golden hamsters.

The cholesterol-lowering activities of oats and barley are commonly attributed to the beta-glucan fractions. Although beta-glucan is present in both grains and appears to be chemically similar, the effect of source on cholesterol-lowering activity has not been evaluated. In the present study, the antiatherogenic properties of beta-glucan concentrates from oats and barley were evaluated in Syrian golden F(1)B hamsters consuming a semipurified hypercholesterolemic diet (HCD) containing cholesterol (0.15 g/100 g), hydrogenated coconut oil (20 g/100 g) and cellulose (15 g/100 g). After a 2-wk lead-in period, control hamsters were fed the HCD, whereas experimental hamsters consumed HCD formulated to include beta-glucan (2, 4, or 8 g/100 g) by addition of beta-glucan concentrate prepared from oats or barley at the expense of cellulose. Compared with control hamsters, dose-dependent decreases that were similar in magnitude in plasma total and LDL cholesterol concentrations were observed in hamsters fed beta-glucan from either source at wk 3, 6 and 9. Compared with controls, liver cholesterol concentrations were also reduced (P < 0.05) in hamsters consuming 8 g/100 g oat or barley beta-glucan. In agreement with previously proposed mechanisms, total fecal neutral sterol concentrations were significantly increased (P < 0.05) in hamsters consuming 8 g/100 g barley or oat beta-glucan. Aortic cholesterol ester concentrations were significantly reduced (P < 0.05) in hamsters fed 8 g/100 g beta-glucan from barley or oats. Although aortic total cholesterol and cholesterol ester concentrations were significantly correlated with LDL cholesterol (r = 0.565, P < 0.004 and r = 0.706, P < 0.0001, respectively), this association could explain only half of the variability. This study demonstrated that the cholesterol-lowering potency of beta-glucan is approximately identical whether its origin was oats or barley.

5alpha, 8alpha-Epidioxysterol sulfate from a diatom Odontella aurita.

A 5alpha,8alpha-epidioxysterol sulfate was isolated from the cultured diatom Odontella aurita (NIES 589), and its structure was elucidated by spectroscopic methods.