A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

The clinical and laboratory investigation of dysbetalipoproteinemia.

Familial dysbetalipoproteinemia (type III hyperlipoproteinemia) is a potentially underdiagnosed inherited dyslipidemia associated with greatly increased risk of coronary and peripheral vascular disease. The mixed hyperlipidemia observed in this disorder usually responds well to appropriate medical therapy and lifestyle modification. Although there are characteristic clinical features such as palmar and tuberous xanthomata, associated with dysbetalipoproteinemia, they are not always present, and their absence cannot be used to exclude the disorder. The routine lipid profile cannot distinguish dysbetalipoproteinemia from other causes of mixed hyperlipidemia and so additional investigations are required for confident diagnosis or exclusion. A range of investigations that have been proposed as potential diagnostic tests are discussed in this review, but the definitive biochemical test for dysbetalipoproteinemia is widely considered to be beta quantification. Beta quantification can determine the presence of "ß-VLDL" in the supernatant following ultracentrifugation and whether the VLDL cholesterol to triglyceride ratio is elevated. Both features are considered hallmarks of the disease. However, beta quantification and other specialist tests are not widely available and are not high-throughput tests that can practically be applied to all patients with mixed hyperlipidemia. Using apolipoprotein B (as a ratio either to total or non-HDL cholesterol or as part of a multi-step algorithm) as an initial test to select patients for further investigation is a promising approach. Several studies have demonstrated a high degree of diagnostic sensitivity and specificity using these approaches and apolipoprotein B is a relatively low-cost test that is widely available on high-throughput platforms. Genetic testing is also important in the diagnosis, but it should be noted that most individuals with an E2/2 genotype do not suffer from remnant hyperlipidemia and around 10% of familial dysbetalipoproteinemia cases are caused by rarer, autosomal dominant mutations in APOE that will only be detected if the gene is fully sequenced. Wider implementation of diagnostic pathways utilizing apo B could lead to more rational use of specialist investigations and more consistent detection of patients with dysbetalipoproteinemia. Without the application of a consistent evidence-based approach to identifying dysbetalipoproteinemia, many cases are likely to remain undiagnosed.

Nano electrospray differential mobility analysis based size-selection of liposomes and very-low density lipoprotein particles for offline hyphenation to MALDI mass spectrometry.

Gas-phase electrophoresis of single-charged analytes (nanoparticles) enables their separation according to the surface-dry particle size (Electrophoretic Mobility Diameter, EMD), which corresponds to the diameter of spherical shaped particles. Employing a nano Electrospray Differential Mobility Analyzer (nES DMA), also known as nES Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA), allows sizing/size-separation and determination of particle-number concentrations. Separations are based on a constant high laminar sheath flow and a tunable, orthogonal electric field enabling scanning of EMDs in the nanometer size range. Additionally, keeping the voltage constant, only nanoparticles of a given EMD pass the instrument and can be collected on corresponding supporting materials for subsequent nanoparticle analyses applying e.g. microscopic, immunologic or spectroscopic techniques. In our proof-of-concept study we now focus for the first time on mass spectrometric (MS) characterization of DMA size-selected material. We carried out size-selection of liposomes, vesicles consisting of a lipid bilayer and an aqueous lumen employed as carriers in e.g. pharmaceutic, cosmetic or nutritional applications. Particles of 85 nm EMD were collected on gold-coated silicon wafers. Subsequently, matrix was applied and Matrix-Assisted Laser Desorption / Ionization (MALDI) MS carried out. However, we not only focused on plain liposomes but also demonstrated the applicability of our approach for very heterogeneous low density lipoprotein (VLDL) particles, a transporter of lipid metabolism. Our novel offline hyphenation of gas-phase electrophoresis (termed nES DMA or nES GEMMA) and MALDI-MS opens the avenue to the molecular characterization of size-select nanoparticles of complex nature.

1H NMR spectroscopy quantifies visibility of lipoproteins, subclasses, and lipids at varied temperatures and pressures.

NMR-based quantification of human lipoprotein (sub)classes is a powerful high-throughput method for medical diagnostics. We evaluated select proton NMR signals of serum lipoproteins for elucidating the physicochemical features and the absolute NMR visibility of their lipids. We separated human lipoproteins of different subclasses by ultracentrifugation and analyzed them by 1H NMR spectroscopy at different temperatures (283-323 K) and pressures (0.1-200 MPa). In parallel, we determined the total lipid content by extraction with chloroform/methanol. The visibility of different lipids in the 1H NMR spectra strongly depends on temperature and pressure: it increases with increasing temperatures but decreases with increasing pressures. Even at 313 K, only part of the lipoprotein is detected quantitatively. In LDL and in HDL subclasses HDL2 and HDL3, only 39%, 62%, and 90% of the total cholesterol and only 73%, 70%, and 87% of the FAs are detected, respectively. The choline head groups show visibilities of 43%, 75%, and 87% for LDL, HDL2, and HDL3, respectively. The description of the NMR visibility of lipid signals requires a minimum model of three different compartments, A, B, and C. The thermodynamic analysis of compartment B leads to melting temperatures between 282 K and 308 K and to enthalpy differences that vary for the different lipoproteins as well as for the reporter groups selected. In summary, we describe differences in NMR visibility of lipoproteins and variations in biophysical responses of functional groups that are crucial for the accuracy of absolute NMR quantification.

Resolution of Lipoprotein Subclasses by Charge Detection Mass Spectrometry.

Lipoproteins are micelle-like assemblies that are key players in the pathogenesis of atherosclerosis. High-density lipoprotein (HDL), low-density lipoproteins (LDL), and very low density lipoprotein (VLDL) are the three major classes present in fasting plasma. Within each class, there is a broad size distribution with wide variations in protein and lipid content. The development of better metrics for cardiovascular risk is thought to depend on better characterization of lipoprotein subclasses. Using charge detection mass spectrometry (CDMS), the mass distributions of HDL, LDL, and VLDL have been directly measured for the first time. In the case of HDL, seven distinct subpopulations were resolved using a two-dimensional correlation of charge and mass. The resolved components are assigned to HDL particles containing different numbers of the key structural proteins apolipoprotein A-I and apolipoprotein A-II.

Alleviating VLDL overproduction is an important mechanism for Laminaria japonica polysaccharide to inhibit atherosclerosis in LDLr-/- mice with diet-induced insulin resistance.

SCOPE: The overproduction of very low density lipoprotein (VLDL) is an important cause for initiation and development of atherosclerosis, which is highly associated with insulin signaling. The aim of this work is to verify whether the inhibition of VLDL overproduction is an underlying mechanism for a Laminaria japonica polysaccharide (LJP61A (where LJP is L. japonica)) to resist atherosclerosis. METHODS AND RESULTS: LJP61A (50 and 200 mg/kg/day) was orally administered to a high-fat diet (HFD)-fed LDL receptor deficient mice for 14 weeks. LJP61A significantly attenuated insulin resistance, hepatic steatosis, atherosclerosis, and dyslipidemia. Meanwhile, LJP61A ameliorated the HFD-induced impairment of hepatic insulin signaling and reduced VLDL overproduction via regulating the expression of genes involved in the assembly and secretion of VLDL. To study the possibility that the inhibition of mammalian target of rapamycin complex 1 and stimulation of Forkhead box protein O1 (Foxo1) nuclear exclusion is a result of LJP61A via regulating insulin signaling, LJP61A was administrated to HepG2 cells in the presence or absence of mTOR inhibitor and Foxo1 inhibitor. Results showed that LJP61A alleviated VLDL overproduction via regulating insulin receptor substrate mediated phosphatidylinositide 3-kinase AKT mammalian target of rapamycin complex 1 and phosphatidylinositide 3-kinase AKT-Foxo1 signaling pathways. CONCLUSION: These results suggested that LJP61A ameliorated HFD-induced insulin resistance to attenuate VLDL overproduction possibly via regulating insulin signaling, leading to the inhibition of atherosclerosis.

Lipoprotein profiling methodology based on determination of apolipoprotein concentration.

AIM: Abnormal lipid metabolism results in the alteration of lipid compositions in lipoproteins; therefore an accurate and quantitative analytical approach is required for the detailed structural characterization of lipoproteins. However, the specific lipid composition of each lipoprotein particle is poorly understood. MATERIALS & METHODS: Lipid composition of very-low-density lipoprotein and low-density lipoprotein particles derived from myocardial infarction-prone rabbits was determined by normalization of lipidomics data using apoB-100 levels. RESULTS: The ratio of lipid levels between very-low-density lipoprotein and low-density lipoprotein particles was different according to not only lipid classes, but also phosphatidylethanolamine subclasses by applying our developed methodology to myocardial infarction-prone rabbits. CONCLUSION: Our novel analytical approach represents to be a potentially useful tool to obtain particle-specific lipid components of lipoproteins.

The postprandial situation as a pro-inflammatory condition / La situación posprandial como condición proinflamatoria

Postprandial lipemia has been associated with cardiovascular disease. The current pathophysiological concept is that postprandial remnant lipoproteins migrate into the subendothelial space and that remnants activate circulating leukocytes and endothelial cells. Activated monocytes adhere to endothelial adhesion molecules, facilitating subendothelial migration of monocytes. These cells differentiate into macrophages, with the risk of foam cell formation, due to uptake of remnants and modified lipoproteins. Evidence is emerging that specific interventions may reduce the atherogenic postprandial inflammation. Fruits rich in polyphenols, virgin olive oil, carotenoids and exercise have recently been found to reduce postprandial inflammation. Pharmaceutical interventions with fibrates or statins not only improve the overall lipid profile, but reduce postprandial inflammation as well. This review will deal with the current concept of postprandial inflammation in relation to the development of atherosclerosis and potential interventions to reduce postprandial inflammation

La lipidemia posprandial está relacionada con la enfermedad cardiovascular. El concepto patofisiológico actual es que las partículas remanentes traspasan el endotelio, activan los leucocitos y las células endoteliales. Los monocitos activados se adhieren a la paredendotelial por mediación de moléculas de adhesión, facilitando así la migración de los monocitos al espacio subendotelial. Estas células se transforman en macrófagos, convirtiéndose definitivamente en células espumosas después de haber internalizado las partículas remanentes y otras lipoproteínas modificadas. Recientes estudios sugieren que existen intervenciones efectivas para modular la inflamación posprandial, y de esta forma rebajar el riesgo cardiovascular. Frutas ricas en polifenoles, aceite de oliva virgen, el caroteno y el ejercicio son ejemplos que han demostrado una reducción de la inflamación posprandial. El tratamiento con estatinas y fibratos no solo mejora el perfil lipídico, sino que también rebaja la lipidemia posprandial. Esta revisión describe los recientes conceptos de la inflamación posprandial relacionada con la generación de ateroesclerosis y también trata las intervenciones que pueden influir positivamente en la inflamación posprandial

A power set-based statistical selection procedure to locate susceptible rare variants associated with complex traits with sequencing data.

MOTIVATION: Existing association methods for rare variants from sequencing data have focused on aggregating variants in a gene or a genetic region because of the fact that analysing individual rare variants is underpowered. However, these existing rare variant detection methods are not able to identify which rare variants in a gene or a genetic region of all variants are associated with the complex diseases or traits. Once phenotypic associations of a gene or a genetic region are identified, the natural next step in the association study with sequencing data is to locate the susceptible rare variants within the gene or the genetic region. RESULTS: In this article, we propose a power set-based statistical selection procedure that is able to identify the locations of the potentially susceptible rare variants within a disease-related gene or a genetic region. The selection performance of the proposed selection procedure was evaluated through simulation studies, where we demonstrated the feasibility and superior power over several comparable existing methods. In particular, the proposed method is able to handle the mixed effects when both risk and protective variants are present in a gene or a genetic region. The proposed selection procedure was also applied to the sequence data on the ANGPTL gene family from the Dallas Heart Study to identify potentially susceptible rare variants within the trait-related genes. AVAILABILITY AND IMPLEMENTATION: An R package 'rvsel' can be downloaded from http://www.columbia.edu/∼sw2206/ and http://statsun.pusan.ac.kr.

Activation of paraoxonase 1 after hemodialysis is associated with HDL remodeling and its increase in the HDL2 fraction and VLDL.

BACKGROUND: Paraoxonase 1 (PON1) activity is lower in renal failure patients. We hypothesize that part of the salutatory effect of hemodialysis on PON1 activity that we have previously found is due to HDL remodeling and shift of PON1 among HDL particles. METHODS: A total of 42 patients (18 females and 24 males, 63 ± 12 yr) on long-term HD, with a mean dialysis course of 6.4 yr (range 1-19 yr), were recruited. PON1 arylesterase and lactonase activities, PON1 and apolipoprotein distribution in HDL subclasses were measured by gel gradient electrophoresis and western blotting. RESULTS: The 3 different activities of PON1 we measured were significantly lower in patients as compared to control subjects; lactonase by 11%, triesterase by 19% and arylesterase by 20%, p<0.01. HDL increased slightly by 4.6%. LDL increased by 13% and VLDL decreased by 30%. These data are compatible with enhanced lipolysis of VLDL that is transformed into LDL. VLDL-PON1 activity increases significantly by 60%. PON1 activity increases by 16% in HDL2 whereas by this approach we could determine a 10% increase in the total area under the curve corresponding to total HDL. Changes in total lactonase activity were associated with changes in VLDL-PON1 and HDL2. In parallel with PON1 activation and shifts among particles, there are significant changes in apoE which increases notably in HDL2, paralleling the changes in PON1. No significant changes in apoAI or apoA-II the main structural HDL apolipoproteins were apparent after dialysis. CONCLUSIONS: HD produces an activation of PON1 that can be predicted in part (30%) by efficiency of dialysis and in part (25%) by PON1 shifts to HDL2or VLDL (p<0.01). The removal of inhibitors and the change in the environment of PON1 in the micro-heterogeneity of HDL subclasses optimizes PON1 activity.

Identification of molecular species of cholesteryl ester hydroperoxides in very low-density and intermediate-density lipoproteins.

BACKGROUND: Oxidation of lipoproteins is thought to play a crucial role in atherogenesis. Role for triglyceride-rich lipoproteins in atherogenesis is unclear. Thus, we aimed to investigate whether cholesteryl ester hydroperoxides (CEOOH) are present in very low-density lipoproteins (VLDL) and intermediate-density lipoproteins (IDL) by using highly sensitive liquid chromatography/mass spectrometry. METHODS: Total lipids were extracted from the plasma of healthy donors (n = 6) and their fractions of VLDL and IDL. Additional three plasma samples were analysed freshly for CEOOH. Detection and identification of CEOOH was conducted by liquid chromatography/LTQ ion trap mass spectrometry/Orbitrap high mass accuracy mass spectrometry. Authentic standards of CEOOH were used for unequivocal identification on the basis of their mass spectra. RESULTS: We identified six molecular CEOOH species overall, namely, Ch18:1-OOH, Ch18:2-OOH, Ch18:3-OOH, Ch20:4-OOH, Ch20:5-OOH and Ch22:6-OOH. Of them, Ch18:2-OOH, Ch20:5-OOH, Ch20:4-OOH and Ch22:6-OOH were detected in all IDL samples, while only Ch20:4-OOH was detected in all VLDL samples. All of CEOOH species except for Ch18:3-OOH were detected in plasma, with constant detection of Ch20:5-OOH, and Ch22:6-OOH in all plasma samples. CONCLUSION: The presence of CEOOH species in VLDL and IDL was confirmed with the analytical sensitivity of 0.1 pmol, showing the constant appearance of more CEOOH species in IDL than VLDL. This finding might add biochemical evidences of atherogenicity of these lipoproteins. Clinical utility of measuring CEOOH level in these lipoproteins need to be investigated for the risk assessment of the cardiovascular disease.

Parameter trajectory analysis to identify treatment effects of pharmacological interventions.

The field of medical systems biology aims to advance understanding of molecular mechanisms that drive disease progression and to translate this knowledge into therapies to effectively treat diseases. A challenging task is the investigation of long-term effects of a (pharmacological) treatment, to establish its applicability and to identify potential side effects. We present a new modeling approach, called Analysis of Dynamic Adaptations in Parameter Trajectories (ADAPT), to analyze the long-term effects of a pharmacological intervention. A concept of time-dependent evolution of model parameters is introduced to study the dynamics of molecular adaptations. The progression of these adaptations is predicted by identifying necessary dynamic changes in the model parameters to describe the transition between experimental data obtained during different stages of the treatment. The trajectories provide insight in the affected underlying biological systems and identify the molecular events that should be studied in more detail to unravel the mechanistic basis of treatment outcome. Modulating effects caused by interactions with the proteome and transcriptome levels, which are often less well understood, can be captured by the time-dependent descriptions of the parameters. ADAPT was employed to identify metabolic adaptations induced upon pharmacological activation of the liver X receptor (LXR), a potential drug target to treat or prevent atherosclerosis. The trajectories were investigated to study the cascade of adaptations. This provided a counter-intuitive insight concerning the function of scavenger receptor class B1 (SR-B1), a receptor that facilitates the hepatic uptake of cholesterol. Although activation of LXR promotes cholesterol efflux and -excretion, our computational analysis showed that the hepatic capacity to clear cholesterol was reduced upon prolonged treatment. This prediction was confirmed experimentally by immunoblotting measurements of SR-B1 in hepatic membranes. Next to the identification of potential unwanted side effects, we demonstrate how ADAPT can be used to design new target interventions to prevent these.

Does a diet high or low in fat influence the oxidation potential of VLDL, LDL and HDL subfractions?

BACKGROUND AND AIMS: High-fat diets have become increasingly popular for weight-loss, but their effect on the oxidation potential of lipoprotein subfractions has not been studied. Therefore, this study compared the effects of high-fat vs. low-fat weight reduction diets on this parameter. METHODS AND RESULTS: Very-low, low- and high-density lipoprotein (VLDL, LDL & HDL) subfractions were isolated by rapid ultracentrifugation from 24-overweight/obese subjects randomised to a high- or low-fat diet. The lipoprotein subfractions were assessed for oxidation potential by measuring conjugated diene (CD) production and time at half maximum. We found a significant between-group difference in oxidation potential. Specifically, a high-fat diet led to increased CD production in VLDL(A-D) and HDL(2&3), and a prolongation of time at half maximum. Within-group differences found that CDs increased in VLDL(A&D), LDL(I-III) and HDL(2&3) in the high-fat group and fell in VLDL(A-C) and HDL(2&3) and increased in LDL(I&II), in the low-fat group. Furthermore, following both diets all lipoprotein subfractions, except LDL(II) in the low-fat group, were protected against oxidation. CONCLUSION: These results demonstrate that at first glance, a high-fat diet may be indicative of having heart-protective properties. However, this may be erroneous, as although the time for oxidation to occur was prolonged, once this occurred these lipoproteins had the potential to produce significantly more oxidised substrate. Conversely, a low-fat diet may be considered anti-atherogenic, as these subfractions were protected against oxidation and mainly contained fewer oxidised substrate. Thus, increased fat intake may, by increasing the oxidation product within lipoprotein subfractions, increase cardiovascular disease.

Hepatotoxicity associated with lapatinib in an experimental rat model.

BACKGROUND: The current study is the first to evaluate the biochemical and histopathological features of hepatic toxicity of lapatinib. METHODS: Twenty Wistar albino rats were allocated into three groups: experimental toxicity was induced with lapatinib (10mg/kg) administered for 28 days (Group 1), 42 days (Group 2) orally in a single dose by gavage. Control group received only sterile water. Rats in Group 1 and Group 2 were sacrificed after the collection of blood and tissue samples on the 28th and 42nd days, respectively. RESULTS: Subjects in Group 1 and Group 2 had significantly higher levels of alanin aminotransferase (ALT), albumin, triglyceride and very low density lipoprotein (VLDL) when compared with the control group. None of the subjects in the two experimental groups showed normal histology. There were parenchymal acinar transformation zones, sinusoidal dilatation, hydropic degeneration of hepatocytes, vacuolisation of hepatocytes around the portal areas, and mild inflammation with dominance of mononuclear cells besides neutrophil and eosinophil leucocytes in portal areas, especially pronounced in Group 2. CONCLUSION: This study demonstrated that lapatinib brings about deterioration of lipid profile and triggers hepatic toxicity mainly as sinusoidal injury with elevation in transaminase levels, especially ALT.

Decreases in the serum VLDL-TG/non-VLDL-TG ratio from early stages of chronic hepatitis C: alterations in TG-rich lipoprotein levels.

BACKGROUND: The liver secretes very-low-density lipoproteins (VLDLs) and plays a key role in lipid metabolism. Plasma total triglyceride (TG) level variations have been studied in patients with hepatitis C virus (HCV)-related chronic hepatitis (CH-C). However, the results of these studies are variable. A homogenous assay protocol was recently proposed to directly measure the TG content in VLDL (VLDL-TG) and VLDL remnants. METHODOLOGY/PRINCIPAL FINDINGS: Using the assay protocol, we determined serum VLDL-TG levels in 69 fasting patients with biopsy-proven HCV-related chronic liver disease and 50 healthy subjects. Patients were classified into stages F0-F4 using the 5-point Desmet scale. Serum total TG levels in patients with non-cirrhotic (F1-F3) CH-C did not demonstrate significant differences compared with healthy subjects, but serum VLDL-TG levels did demonstrate significant differences. Mean serum VLDL-TG levels tended to decrease with disease progression from F1 to F4 (cirrhosis). Compared with healthy subjects, serum non-VLDL-TG levels significantly increased in patients with stages F2 and F3 CH-C; however, we observed no significant difference in patients with liver cirrhosis. Furthermore, the serum VLDL-TG/non-VLDL-TG ratio, when taken, demonstrated a significant decrease in patients with CH-C from the mildest stage F1 onward. CONCLUSIONS/SIGNIFICANCE: The decrease in serum VLDL-TG levels was attenuated by increase in non-VLDL-TG levels in patients with non-cirrhotic CH-C, resulting in comparable total TG levels. Results of previous studies though variable, were confirmed to have a logical basis. The decrease in the serum VLDL-TG/non-VLDL-TG ratio as early as stage F1 demonstrated TG metabolic alterations in early stages of CH-C for the first time. The involvement of TG metabolism in CH-C pathogenesis has been established in experimental animals, while conventional TG measurements are generally considered as poor indicators of CH-C progression in clinical practice. The serum VLDL-TG/non-VLDL-TG ratio, which focuses on TG metabolic alterations, may be an early indicator of CH-C.

Effects of palmitic acid on lipid metabolism homeostasis and apoptosis in goose primary hepatocytes.

Studies have shown that not only does palmitic acid promote triglyceride (TG) accumulation, but it also affects cell viability in in vitro steatosis models. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effects of palmitic acid on the lipid metabolism homeostasis pathway and on apoptosis were determined. The authors measured the mRNA levels of genes involved in TG synthesis, lipid deposition, fatty acid oxidation and the assembly and secretion of VLDL-TG in goose primary hepatocytes. The results indicated that palmitic acid can significantly reduce the activity of goose hepatocytes, and that palmitic acid had a significant effect on TG accumulation; however, with increasing palmitic acid concentrations, the extracellular TG and extracellular VLDL concentration gradually decreased. With increasing palmitic acid concentrations, the gene expression levels of DGAT1, DGAT2, PPARα, CPT-1, FoxO1 and MTTP (which regulate hepatic TG synthesis, fatty acid oxidation and the assembly and secretion of VLDL-TGs) first increased and then decreased; the change in PLIN gene expression was palmitic acid dose-dependent, similar to the regulatory mode of intracellular TG accumulation. In conclusion, this study clearly shows that palmitic acid can promote TG accumulation and induce apoptosis in goose primary hepatocytes, and this effect may be related to the lipid metabolism pathway.

Intravascular transfer contributes to postprandial increase in numbers of very-low-density hepatitis C virus particles.

BACKGROUND & AIMS: The physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection. METHODS: Fasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially. RESULTS: The amount of HCV RNA in the VLDF (HCV(VLDF)) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCV(VLDF) rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B-100 and not apolipoprotein B-48, showed that HCV(VLDF) is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCV(VLDF), compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCV(VLDF). CONCLUSIONS: Dietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCV(VLDF) indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.

Proteomic analysis of the triglyceride-rich lipoprotein-laden foam cells.

In hypertriglyceridaemic individuals, atherosclerogenesis is associated with the increased concentrations of very low density lipoprotein (VLDL) and VLDL-associated remnant particles. In vitro studies have suggested that VLDL induces foam cells formation. To reveal the changes of the proteins expression in the process of foam cells formation induced by VLDL, we performed a proteomic analysis of the foam cells based on the stimulation of differentiated THP-1 cells with VLDL. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, 14 differentially expressed proteins, containing 8 up-regulated proteins and 6 down-regulated proteins were identified. The proteins are involved in energy metabolism, oxidative stress, cell growth, differentiation and apoptosis, such as adipose differentiation-related protein (ADRP), enolase, S100A11, heat shock protein 27 and so on. In addition, the expression of some selected proteins was confirmed by Western blot and RT-PCR analysis. The results suggest that VLDL not only induces lipid accumulation, but also brings about foam cells diverse characteristics by altering the expression of various proteins.

Intrahepatic fat, not visceral fat, is linked with metabolic complications of obesity.

Visceral adipose tissue (VAT) is an important risk factor for obesity-related metabolic disorders. Therefore, a reduction in VAT has become a key goal in obesity management. However, VAT is correlated with intrahepatic triglyceride (IHTG) content, so it is possible that IHTG, not VAT, is a better marker of metabolic disease. We determined the independent association of IHTG and VAT to metabolic function, by evaluating groups of obese subjects, who differed in IHTG content (high or normal) but matched on VAT volume or differed in VAT volume (high or low) but matched on IHTG content. Stable isotope tracer techniques and the euglycemic-hyperinsulinemic clamp procedure were used to assess insulin sensitivity and very-low-density lipoprotein-triglyceride (VLDL-TG) secretion rate. Tissue biopsies were obtained to evaluate cellular factors involved in ectopic triglyceride accumulation. Hepatic, adipose tissue and muscle insulin sensitivity were 41, 13, and 36% lower (P < 0.01), whereas VLDL-triglyceride secretion rate was almost double (P < 0.001), in subjects with higher than normal IHTG content, matched on VAT. No differences in insulin sensitivity or VLDL-TG secretion were observed between subjects with different VAT volumes, matched on IHTG content. Adipose tissue CD36 expression was lower (P < 0.05), whereas skeletal muscle CD36 expression was higher (P < 0.05), in subjects with higher than normal IHTG. These data demonstrate that IHTG, not VAT, is a better marker of the metabolic derangements associated with obesity. Furthermore, alterations in tissue fatty acid transport could be involved in the pathogenesis of ectopic triglyceride accumulation by redirecting plasma fatty acid uptake from adipose tissue toward other tissues.

FT-IR spectroscopy of lipoproteins--a comparative study.

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.