Synthesis of Amphiphilic Hepatocyte Targeting Cholesterylated Thiogalactosides

Six amphiphilic cholesterylated thiogalactosides la-f with high affinity for the asialoglycoprotein receptor have been synthesized by coupling 2,3,4,6-tetra -O-acetyl-l-thio-β-D-galactopyranose 8 with prepared cholesterol derivatives 7a-f, then by deacetylating. Preliminary results show liposomes containing those galactosides derivatives exhibited higher affinity and transfection activity in hepatoma cells HepG2 and SMMC-7721.

Pathogenic contribution of cholesteryl ester accumulation in the brain to neurodegenerative disorders

Cholesteryl esters(CEs) have been increasingly implicated in neurodegenerative disorders such as Alzheimer's disease(AD).Alois Alzheimer noted three prominent neuropathologic features in his original analysis of the AD brain:senile plaques,neurofibrillary tangles,and lipid granule accumulation.Senile plaques,which are aggregates of amyloid-beta(Aβ),and neurofibrillary tangles,which are aggregates of phosphorylated tau,have been regarded as more consistent characteristics of the AD brain compared with lipid granule accumulation and thus have been studied more intensively(Foley,2010).

Synthesis of a novel tri-antennary galactoside with high hepatocyte targeting

A novel bifunctional compound carrying duster thiogalactoside as the cell targeting ligands was synthesized for gene delivery to hepatocytes. Tetra-antennary dendr-OMs4 5 was used as a scaffold for the attachment of three galactosides, while the other mesylate end was linked with cholesterol through poly（ethylene glycol） chain. This design provided an effective entry for the synthesis of the bifunctional compound.

Enhancement of human ACAT1 gene expression to promote the macrophage-derived foam cell formation by dexamethasone

In macrophages, the accumulation of cholesteryl esters synthesized by the activated acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) results in the foam cell formation, a hallmark of early atherosclerotic lesions. In this study, with the treatment of a glucocorticoid hormone dexamethasone (Dex), lipid staining results clearly showed the large accumulation of lipid droplets containing cholesteryl esters in THP-1-derived macrophages exposed to lower concentration of the oxidized low-density lipoprotein (ox-LDL). More notably, when treated together with specific anti-ACAT inhibitors, the abundant cholesteryl ester accumulation was markedly diminished in THP-1-derived macrophages, confirming that ACAT is the key enzyme responsible for intracellular cholesteryl ester synthesis. RT-PCR and Western blot results indicated that Dex caused up-regulation of human ACAT1 expression at both the mRNA and protein levels in THP-1 and THP-1-derived macrophages. The luciferase activity assay demonstrated that Dex could enhance the activity of human ACAT1 gene P1 promoter, a major factor leading to the ACAT1 activation, in a cell-specific manner. Further experimental evidences showed that a glucocorticoid response element (GRE) located within human ACAT1 gene P1 promoter to response to the elevation of human ACAT1 gene expression by Dex could be functionally bound with glucocorticoid receptor (GR) proteins. These data supported the hypothesis that the clinical treatment with Dex, which increased the incidence of atherosclerosis, may in part due to enhancing the ACAT1 expression to promote the accumulation of cholesteryl esters during the macrophage-derived foam cell formation, an early stage of atherosclerosis.

Genetic variants involved in gallstone formation and capsaicin metabolism,and the risk of gallbladder cancer in Chilean women

AIM:To determine the effects of genetic variants associated with gallstone formation and capsaicin (a pungent component of chili pepper) metabolism on the risk of gallbladder cancer (GBC).METHODS: A total of 57 patients with GBC, 119 patients with gallstones, and 70 controls were enrolled in this study. DNA was extracted from their blood or paraffi n block sample using standard commercial kits. The statuses of the genetic variants were assayed using Taqman SNP Genotyping Assays or Custom Taqman SNP Genotyping Assays.RESULTS:The non-ancestral T/T genotype of apolipoprotein B rs693 polymorphism was associated with a decreased risk of GBC (OR:0.14,95% CI:0.03-0.63). The T/T genotype of cholesteryl ester transfer protein (CETP) rs708272 polymorphism was associated with an increased risk of GBC (OR:5.04,95% CI:1.43-17.8).CONCLUSION: Genetic variants involved in gallstone formation such as the apolipoprotein B rs693 and CETP rs 708272 polymorphisms may be related to the risk of developing GBC in Chilean women.

Prevention of fatal hepatic complication in schistosomiasis by inhibition of CETP

Schistosoma japonicum, once endemic all the East Asia, remains as a serious public health problem in certain regions. Ectopic egg embryonation in the liver causes granulomatosis and eventually fatal cirrhosis, so that prevention of this process is one of the keys to reduce its mortality. The embryonation requires cholesteryl ester from HDL of the host blood for egg yolk formation, and this reaction is impaired from the abnormal large HDL in genetic cholesteryl ester transfer protein （CETP） deficiency. When CETP was expressed in mice that otherwise lack this protein, granulomatosis of the liver was shown increased compared to the wild type upon infec- tion of Schistosoma japonicum. The CETP deficiencies accumulated exclusively in East Asia, from Indochina to Siberia, so that Shistosomiasis can be a screening factor for this accumulation. CD36 related protein （CD36RP） was identified as a protein for this reaction, cloned from the cDNA library of Schistosoma japonicum with 1880-bp encoding 506 amino acids. The antibody against the extracellular loop of CD36RP inhibited cholesteryl ester uptake from HDL and suppressed egg embryonation in culture. Therefore, inhibition of CETP is a potential approach to prevent liver granulomatosis and thereby fatal liver cirrhosis in the infection of Schistosoma japonicum.

An intravenous clarithromycin lipid emulsion with a high drug loading, H-bonding and a hydrogen-bonded ion pair complex exhibiting excellent antibacterial activity

The aim of this study was to develop an intravenous clarithromycin lipid emulsion(CLE)with good stability and excellent antibacterial activity. The CLE was prepared by the thinfilm dispersed homogenization method. The interaction between clarithromycin(CLA) and cholesteryl hemisuccinate(CHEMS) was confirmed by DSC, FT-IR and^1H NMR analysis. The interfacial drug loading, thermal sterilization, freeze–thaw stability, and in vitro and in vivo antibacterial activity were investigated systematically. DSC, FT-IR and^1H NMR spectra showed that CHEMS(CLA: CHEMS, M ratio 1:2) could interact with CLA through H-bonding and a hydrogen-bonded ion pair. The CHEMS was found necessary to maintain the stability of CLE.Ultracentrifugation showed that almost 88% CLA could be loaded into the interfacial layer.The optimized CLE formulation could withstand autoclaving at 121 °C for 10 min and remain stable after three freeze–thaw cycles. The in vitro susceptibility test revealed that the CLA–CHEMS ion-pair and CLE have similar activity to the parent drug against many different bacterial strains. The in vivo antibacterial activity showed that the ED50 of intravenous CLE was markedly lower than that of CLA solution administrated orally. CLE exhibited pronounced antibacterial activity and might be a candidate for a new nanocarrier for CLA with potential advantages over the current commercial formulation.

High density lipoprotein and cardiovascular diseases

Several epidemiological studies have clearly shown that low plasma levels of high density lipoprotein cholesterol (HDL-C) represent a cardiovascular disease (CVD) risk factor. However, it is unclear if there is a causal association between HDL-C concentration and CVD. A recent study published in the Lancet, which performed two Mendelian randomization analyses, showed that increased HDL-C levels were not associated with a decreased risk of myocardial infarction. These findings, together with the termination of the niacin-based AIM-HIGH trial and the discontinuation of cholesteryl ester transfer protein inhibitor dalcetrapib, challenge the concept that raising of plasma HDL-C will uniformly translate into reductions in CVD risk. HDL particles exhibit several anti-atherosclerotic properties, such as anti-inflammatory and anti-oxidative activities and cellular cholesterol efflux activity. Furthermore, HDL particles are very heterogeneous in terms of size, structure, composition and metabolism. HDL functionality may be associated more strongly with CVD risk than the traditional HDL-C levels. More research is needed to assess the association of the structure of HDL particle with its functionality and metabolism.

Effect of Tumor Necrosis Factor-α on Acyl Coenzyme A: Cholesteryl Acyltransferase Activity and ACAT1 Gene Expression in THP-1 Macrophages

In order to explore the effect and mechanisms of tumor necrosis factor-α （TNF-α） on the activity of the acyl coenzyme A： cholesteryl acyltransferase （ACAT）, THP-I monocytes were cul- tured and induced to differentiate into macrophages with phorbol ester. TNF-α （60 ng/mL） was added at different time points into the macrophage-containing medium and the ACAT enzyme activity was measured by quantifying the incorporation of [1-^14C] oleoyl CoA into cholesteryl esters. The expression of ACAT-1 protein and mRNA was respectively detected by Western blotting and RT-PCR in THP-1 macrophages 24 h after treatment with TNF-α （60 ng/mL）. The results indicated that ACAT activity in THP-I macrophages treated with TNF-α was increased in a time-dependent manner. The expression levels of ACAT-1 protein and mRNA were significantly increased in THP-I macrophages after treatment with TNF-α （P〈0.05）. It was suggested that TNF-α could increase the activity of ACAT in THP-1 macrophages by up-regulating the expression of ACAT-1 gene.

Natural history and management of liver dysfunction in lysosomal storage disorders

Lysosomal storage disorders(LSD)are a rare group of genetic disorders.The major LSDs that cause liver dysfunction are disorders of sphingolipid lipid storage[Gaucher disease(GD)and Niemann-Pick disease]and lysosomal acid lipase deficiency[cholesteryl ester storage disease and Wolman disease(WD)].These diseases can cause significant liver problems ranging from asymptomatic hepatomegaly to cirrhosis and portal hypertension.Abnormal storage cells initiate hepatic fibrosis in sphingolipid disorders.Dyslipidemia causes micronodular cirrhosis in lipid storage disorders.These disorders must be keenly differentiated from other chronic liver diseases and non-alcoholic steatohepatitis that affect children and young adults.GD,Niemann-Pick type C,and WD also cause neonatal cholestasis and infantile liver failure.Genotype and liver phenotype correlation is variable in these conditions.Patients with LSD may survive up to 4-5 decades except for those with neonatal onset disease.The diagnosis of all LSD is based on enzymatic activity,tissue histology,and genetic testing.Enzyme replacement is possible in GD and Niemann-Pick types A and B though there are major limitations in the outcome.Those that progress invariably require liver transplantation with variable outcomes.The prognosis of Niemann-Pick type C and WD is universally poor.Enzyme replacement therapy has a promising role in cholesteryl ester storage disease.This review attempts to outline the natural history of these disorders from a hepatologist’s perspective to increase awareness and facilitate better management of these rare disorders.

The effects of fucoidans from Laminaria japonica on AAPH mediated oxidation of human low-density lipoprotein

Five fucoidan fractions from Laminaria japonica with different sulfate content and molecular weight were prepared by anion-exchange chromatography and mild acid hydrolysis. Their antioxidant effects on azo radicals 2-2＇-Azobis （2-amidinopropane） dihydrochloride （AAPH） induced oxidation of human low-density lipoprotein （LDL） were evaluated by monitoring cholesteryl ester hydroperoxides （CHL-OOH） formation kinetics through reverse-phase high performance liquid chromatography （RP-HPLC） analysis. Fucoidan F - C with a low molecular mass of 2 000 ～8 000 and a sulfate content of 24.3% had much stronger protective antioxidant effect than other four fucoidan fractions on the hydrophilic radical AAPH induced LDL oxidation. However, the highly sulfated fucoidan fraction L - B with a molecular mass of 20 000 was completely ineffective in protecting LDL from the AAPH induced oxidation. The results suggested that both molecular mass and sulfate content of fucoidan played very important roles in their effects on the AAPH induced LDL oxidation.

Novel LIPA mutations in Mexican siblings with lysosomal acid lipase deficiency

Lysosomal acid lipase(LAL) deficiency is an underrecognized lysosomal disease caused by deficient enzymatic activity of LAL.In this report we describe two affected female Mexican siblings with early hepatic complications.At two months of age,the first sibling presented with alternating episodes of diarrhea and constipation,and later with hepatomegaly,elevated transaminases,high levels of total and low-density lipoprotein cholesterol,and low levels of highdensity lipoprotein.Portal hypertension and grade 2 esophageal varices were detected at four years of age.The second sibling presented with hepatomegaly,elevated transaminases and mildly elevated lowdensity lipoprotein and low high-density lipoprotein at six months of age.LAL activity was deficient in both patients.Sequencing of LIPA revealed two previously unreported heterozygous mutations in exon 4:c.253C>A and c.294C>G.These cases highlight the clinical continuum between the so-called Wolman disease and cholesteryl ester storage disease,and underscore that LAL deficiency represents a single disease with a degree of clinical heterogeneity.

High level of serum cholesteryl ester transfer protein inactive hepatitis C virus infection

AIM: To determine the significance of cholesteryl ester transfer protein(CETP) in lipoprotein abnormalities in chronic hepatitis C virus(HCV) infection.METHODS: We evaluated the significance of the serum concentration of CETP in 110 Japanese patients with chronic HCV infection. Fifty-five patients had active HCV infection, and HCV eradication had been achieved in 55. The role of CETP in serum lipoprotein abnormalities, specifically, in triglyceride(TG) concentrations in the four major classes of lipoproteins, was investigated using Pearson correlations in conjunction with multiple regression analysis and compared them between those with active HCV infection and those in whom eradication had been achieved. RESULTS: The serum CETP levels of patients with active HCV infection were significantly higher than those of patients in whom HCV eradication was achieved(mean ± SD, 2.84 ± 0.69 μg/m L vs 2.40 ± 1.00 μg/m L, P = 0.008). In multiple regression analysis, HCV infection status(active or eradicated) was an independent factor significantly associated with the serum CETP level. TG concentrations in low-density lipoprotein(mean ± SD, 36.25 ± 15.28 μg/m L vs 28.14 ± 9.94 μg/m L, P = 0.001) and high-density lipoprotein(HDL)(mean ± SD, 25.9 ± 7.34 μg/m L vs 17.17 ± 4.82 μg/m L, P < 0.001) were significantly higher in patientswith active HCV infection than in those in whom HCV eradication was achieved. The CETP level was strongly correlated with HDL-TG in patients with active HCV infection(R = 0.557, P < 0.001), whereas CETP was not correlated with HDL-TG in patients in whom HCV eradication was achieved(R =-0.079, P = 0.56). CONCLUSION: Our results indicate that CETP plays a role in abnormalities of lipoprotein metabolism in patients with chronic HCV infection.

Synthesis of Novel Lipids Bearing Cholesteryl Group as Gene Vectors

Some cationic and neutral lipids bearing cholesteryl group were synthesized as gene vectors, and the structures of the compounds were characterized by IR, (HNMR)-H-1, MS and elemental analysis.

Qualitative Distribution of Endogenous Cholesteryl Esters in Plasma of Humans and Three Rodent Species Using Stepwise UPLC-Q-Exactive-MS

Objective:Cholesteryl esters(CEs)are composed of various fatty acyl chains attached to the hydroxyl groups of cholesterol,and abnormalities in their metabolism are related to many diseases.This study aimed to develop an ultrahigh-performance liquid chromatography-quadrupole exactive mass spectrometry(UPLC-Q-Exactive MS)method to identify the CEs in plasma.Methods:First,the MS fragmentation patterns were investigated using seven commercial CE standards.Then,the CEs in plasma were characterized through the accurate mass data of precursor ions and characteristic product ions.A strategy of step-by-step m/z scans in a narrow range was proposed to identify more trace CEs by the full-scan data-dependent MS/MS(ddMS2)mode.Results:A total of 50 CE species consisting of 55 regioisomers were identified in human plasma.Among them,two species were reported for the first time.Conclusion:This study is the most comprehensive identification of CE species in human plasma to date.These results will contribute to the in-depth profiling of CEs in human plasma and provide guidance for animal model selection when studying lipid-related diseases.

Tag single nucleotide polymorphism rs1532624 located in cholesteryl ester transfer protein gene is associated with atherosclerosis cerebral ischemia

Objective: To investigate the relationship between polymorphisms of rs1532624 and rs289741 loci in cholesteryl ester transfer protein(CETP) genes and atherosclerotic cerebral infarction(ACI). Methods: The CETP gene rs1532624 and rs289741 in 95 patients with ACI and 177 healthy subjects were genotyped by Mass ARRAY mass spectrometry. Each locus genotype and allele frequency distributions were compared. Results: The difference of allele frequency distribution between the rs1532624(χ~2=1.723, P=0.189) and rs289741(χ~2=2.466, P=0.116) were not statistically significant. The frequency distribution of rs1532624 genotype between the cerebral infarction group and healthy control group was statistically significant(χ~2=7.096, P=0.029), while rs289741 genotype frequency distribution between the two groups was not statistically significant(χ~2=2.906, P=0.234). Conclusion: ACI have a positive correlation with rs1532624 polymorphism, and AA genotype may be susceptible factors of ACI.

A Convenient Method for the Stereoselective Synthesis of 7αHydroxycholesterol

7α-Hydroxycholesterol was prepared from cholesteryl acetate via allylic browhnation followed by hydroxylation in 73% overall yield.

SPECTROSCOPIC DETERMINATION OF THE AVERAGE NUMBER OF COAGGREGATES (Nco) OF CHOLESTERYL ESTERS AND THE FLUORESCENCE PROBE

Average aggregate number of coaggregates(N_co)of CE-n or BL-n and the fluoresc- ence probe(Np-16)have been determined by using time-resolved fluorescence spectroscopy. Chain-length,hydroxy-group and chain-foldability effects on the N_co have been discussed.

Synthesis and Characterization of Mesomorphic Behaviour of New Mesogenic Compounds Incorporating Cholesteryl Ester Moiety Connected to 1,3,4-Oxadiazole

The synthesis and characterization of new series of liquid crystals containing cholesteryl moiety connected via an ester linkage to 1,3,4-oxadiazole are reported. The molecular structures of the intermediates and target compounds were confirmed by elemental analyses and FT-IR, ^1H NMR and mass spectrometry. The mesomorphic behaviors were investigated by polarizing optical microscopy （POM） attached with hot stage and differential scanning calorimetry （DSC）. Enantiotropic smectic 1, smectic A, blue phase and cholesteric mesophases are exhibited by the newly synthesized compounds.

The Conditions for Se-Enzymes, NO, cGMP and LDL to be Formed and How They are Connected with Each Other

Transportation of energy and substances is fundamental for the conditions of life. The energy from metabolised food is yielded at the reduction of oxygen by the assistance of Se-enzymes. Activated Se-enzymes contain an electron conducting Se-link with elemental Se bounded to Se-cys （Se-cystein） that prevents electron transferring （autoxidable） agents, as arginine, from oxidations. NO is formed from e.g. arginine during the passive state of a Se-enzyme. With NO, instead of O2, bounded to a hem group, destroying oxidations are avoided. This is the role of NO. A nerve signal is activated by the GTP （guanosine triphosphate）-induced triggered transformation of cGMP （cyclic guanosine monophosphate） into GMP, closing the sodium channels. When GTP is consumed, the opposite reaction takes place. The direct influence of the GMP/cGMP quote on a nerve signal makes GMP/cGMP equal to EDHF （endothelium-derived hyperpolarizing factor）. Part of the energy from food is stored and transported in the form of acetyl groups, building up many important molecules of which LDL （low density lipoprotein） is one, containing cholesteryl esters. These are brought in to the cell, decomposed to acetyl groups generating hydrogen, H, making LDL to an essential substance. High levels are connected to impaired energy yielding reactions, perhaps related to low levels of some substances, especially to one or both forms of Se. The necessity of a Se-link and NO induced protection against oxidations by reducible oxygen can no longer be neglected.