Lipoproteins modulate growth and differentiation of cultured human epidermal keratinocytes

The effects of various lipoproteins on the growth and the differentiation of cultured normal human keratinocytes were investigated. Primary cultures of human epidermal keratinocytes were obtained from neonatal foreskin, and then added with lipoproteins, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). Cell growth potential was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. VLDL and LDL enhanced keratinocytes growth and LDL receptor expression at the plasma membrane level. These effects were more remarkably observed in cells cultured with VLDL than in cells cultured with LDL. Apolipoprotein E (ApoE) was highly increased in VLDL treated cells. These results suggest that VLDL binds with high affinity to cell surface receptors and induces cell proliferation.     

Simultaneous Determination of High-Density Lipoprotein,Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10–800, 10–800, 40–1,000 and 20–800 μg L⁻¹, and their limits of detection were 5, 5, 15 and 8 μg L⁻¹, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8–7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.

     

Simultaneous Determination of High-Density Lipoprotein, Very Low-Density Lipoprotein and Low-Density Lipoprotein Subclass in Human Serum by Microchip CE

Lipoproteins, especially high-density lipoproteins (HDL), very low-density lipoproteins (VLDL) and small, dense low-density lipoprotein (sdLDL), are believed to play an important role in the development of atherosclerosis. In this work, a simple, selective and sensitive method for the simultaneous monitoring of these lipoproteins in human serum using microchip capillary electrophoresis was developed. Gold nanoparticles were used as an additive to the running buffer to obtain the absolute separation of the lipoproteins. Under optimised conditions, the linear ranges of large buoyant low-density lipoproteins, sdLDL, VLDL and HDL were 10?C800, 10?C800, 40?C1,000 and 20?C800 ??g L^?1, and their limits of detection were 5, 5, 15 and 8 ??g L^?1, respectively. The intraassay and interassay relative standard deviation of lipoprotein peak areas were in the range of 3.8?C7.4%. For practical application, variations in the serum lipoprotein of coronary heart disease patients were monitored by microchip-based CE. The results showed that the method was applicable for routine clinical use and allowed the rapid detection of different lipoprotein classes as well as their subclasses, thus greatly improving the analysis of atherosclerotic risk factors.     

Analytical capillary isotachophoresis: a routine technique for the analysis of lipoproteins and lipoprotein subfractions in whole serum

A capillary isotachophoretic separation technique was developed for lipoproteins in native serum which, compared with previous electrophoretic techniques, has negligible molecular sieve effects, does not need gel casting, is suitable for whole serum and has a high discriminative power for lipoprotein subfractions. The technique is based on pre-staining whole serum lipoproteins for 30 min at 4 degrees C before separation of 0.5 microliter of the sample in a free-flow capillary system (0.5 mm I.D.) with discontinuous buffer system. In normolipidaemic sera, high-density (HDL) and low-density lipoproteins (VLDL) are separated into two major subpopulations according to their net electric mobility. The identification of these fractions was confirmed by substitution with ultracentrifugally isolated lipoproteins and by their complete absence from Tangier and abetalipoproteinaemic serum. Triglyceride-rich very low-density lipoproteins (VLDL) revealed a defined zone between the HDL and LDL subpopulations. Our preliminary results indicate that the separation of human whole serum lipoproteins by capillary isotachophoresis is a promising method for the determination of lipoprotein subfractions.     

Analysis of lipoproteins with analytical capillary isotachophoresis

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary iso tachophoresis. In addition to patient analysis we investigated the influence of lipid lowering drugs on the lipoprotein subfraction distribution during therapy with analytical capillary isotachophoresis.     

Hypocholesterolemic Effect of Blackcurrant (Ribes nigrum) Extract in Healthy Female Subjects: A Pilot Study

Blackcurrant extract (BCE) ameliorates dyslipidemia in menopausal model animals and in elderly women at a risk of dyslipidemia. However, it is unknown whether the daily intake of BCE can prevent lipid abnormalities in healthy individuals. Lipids are essential for the body, but they also cause arteriosclerosis. In this noncomparative pilot study, we examined the effects of BCE administered for 29 days on serum lipids in young healthy women. Blood samples were collected before and on days 4 and 29 after BCE intake, and 20 lipoprotein fractions in the serum were separated using a gel-permeation high-performance liquid chromatography method to measure the triacylglycerol and cholesterol levels in lipoproteins. There were no effects on lipids on day 4 of BCE intake, but the total cholesterol level decreased on day 29. Furthermore, the levels of total very-low-density lipoprotein (VLDL) cholesterol, small VLDL cholesterol, and large low-density lipoprotein cholesterol were significantly decreased. These results suggest that the daily intake of BCE has a hypocholesterolemic effect in healthy women, and that it is effective in preventing atherosclerosis.     

Analytical determination of polyphenols in olive oils

The increasing popularity of olive oil is mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles, and its richness in phenolic compounds, which act as natural antioxidants and may contribute to the prevention of human disease. An overview of analytical methods for the measurement of polyphenols in olive oil is presented. In principle, the analytical procedure for the determination of individual phenolic compounds in virgin olive oil involves three basic steps: extraction from the oil sample, analytical separation, and quantification. A great number of procedures for the isolation of the polar phenolic fraction of virgin olive oil, utilizing two basic extraction techniques, LLE or SPE, have been included. The reviewed techniques are those based on spectrophotometric methods, as well as analytical separation (gas chromatography (GC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE)). Many reports in the literature determine the total amount of phenolic compounds in olive oils by spectrophometric analysis and characterize their phenolic patterns by capillary gas chromatography (CGC) and, mainly, by reverse phase high-performance liquid chromatography (RP-HPLC); however, CE has recently been applied to the analysis of phenolic compound of olive oil and has opened up great expectations, especially because of the higher resolution, reduced sample volume, and analysis duration. CE might represent a good compromise between analysis time and satisfactory characterization for some classes of phenolic compounds of virgin olive oils.     

Determination of inorganic cations and anions by ion-exchange chromatography with evaporative light-scattering detection

Evaporative light-scattering detection (ELSD) was investigated for the direct determination of alkali and alkaline-earth cations by cation-exchange chromatography. Successful single run analysis of Na+, K+, Mg2+ and Ca2+ was achieved in 11 min on the Hamilton PRP-X200 column using an aqueous solution of ammonium formate as mobile phase under a salt concentration step gradient mode (20 mM and 100 mM). Surprisingly the use of ELSD reveals a weak retention of inorganic anions (Cl-, NO3-, SO4(2-)) onto the polymeric cation exchanger, which enables the simultaneous determination of inorganic anions (C1- and NO3-) associated with the cations analysed (Na+ and K+).     

Isolation of plasma lipoproteins by a combination of differential and density gradient ultracentrifugation

Summary A method for preparative isolation of serum lipoproteins by a combination of differential and density gradient ultracentrifugation is presented. Total plasma lipoproteins are first isolated in a concentrated form by ultracentrifugation in a fixed angle rotor at a plasma background density of 1.21 kg/l. Subsequently, the various lipoprotein classes are separated by density gradient ultracentrifugation in a swinging bucket rotor. The procedure requires only two ultracentrifugation steps and combines advantages of both ultracentrifugation techniques.

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Isolierung von Plasmalipoproteinen durch eine Kombination von Differential- und Dichtegradient-Ultrazentrifugation

Abbreviations VLDL very low density lipoproteins - LDL low density lipoproteins - HDL high density lipoproteins - VHDL very high density lipoproteins     

Comparative study of the determination of triacylglycerol in vegetable oils using chromatographic techniques

The triacylglycerols of some vegetable oil samples were determined using isocratic HPLC with refractive index (RI) detection, gradient solvent HPLC with evaporative light scattering detection (ELSD), capillary GC and theoretical calculations from FAME analysis in order to establish the suitability of these techniques. The response factors and the repeatability were investigated. Generally, the HPLC-RI detection technique can be used without application of response factors. HPLC-ELSD yields inaccurate results for low concentrations. Calculations assuming a 1,3-random 2-random distribution of fatty acids gave good results for olive oil and acceptable results for sunflower oil. The GC analysis requires the use of response factors.     

Microanalytical systems for separations of stratum corneum ceramides

The small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Sil) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 microL/min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH3OH/CHCl3 70:30 v/v to CHCl3 at 2%/min with a flow rate at 5 microL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm2 area of the forearm with 25 mL of ethanol.     

Simultaneous determination of free and total glycerol in biodiesel by capillary electrophoresis using multiple short‐end injection

A rapid method for the simultaneous determination of free glycerol (FG) and total glycerol (TG) in biodiesel by CE using a short‐end multiple injection (SE/MI) configuration system is described. The sample preparation for FG involves the extraction of glycerol with water and for TG a saponification reaction is carried out followed by extraction as in the case of FG. The glycerol extracted in both cases is submitted to periodate oxidation and the iodate ions formed are measured on a CE‐SE/MI system. The relevance of this study lies in the fact that no analytical procedure has been previously reported for the determination of TG (or of FG and TG simultaneously) by CE. The optimum conditions for the saponification/extraction process were 1.25% KOH and 25°C, with a time of only 5 min, and biodiesel mass in the range of 50.0–200.0 mg can be used. Multiple injections were performed hydrodynamically with negative pressure as follows: 50 mbar/3s (FG sample); 50 mbar/6s (electrolyte spacer); 50 mbar/3s (TG sample). The linear range obtained was 1.55–46.5 mg/L with R²> 0.99. The LOD and LOQ were 0.16 mg/L and 0.47 mg/L, respectively for TG. The method provides acceptable throughput for application in quality control and monitoring biodiesel synthesis process. In addition, it offers simple sample preparation (saponification process), it can be applied to a variety biodiesel samples (soybean, castor, and waste cooking oils) and it can be used for the determination of two key parameters related to the biodiesel quality with a fast separation (less than 30 s) using an optimized CE‐SE/MI system.     

Serum amyloid A protein in very low density--and high density lipoproteins during the course of acute myocardial infarction

In a preceding paper we reported on the detection and characterization of human serum amyloid A protein (SAA) in very low density lipoproteins (VLDL) and high density lipoproteins (HDL) of patients after acute myocardial infarction. Here we describe the time course of the occurrence of SAA in VLDL and HDL in the postinfarction period. SAA reached its maximum in VLDL and HDL approximately 53 h after the acute event. At the peak of the acute-phase response, SAA comprised as much as 38% of the total apoproteins of VLDL and HDL. SAA appeared at the same points in time and with nearly the same concentrations in VLDL and HDL. We conclude that SAA is not exchanged in plasma between lipoproteins of different densities and that this protein is secreted on its own by hepatocytes and not as a part of an already constituted lipoprotein particle.     

Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS)

Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity.     

Composition,structure and substrate properties of reconstituted discoidal HDL with apolipoprotein A-I and cholesteryl ester

To investigate the influence of lipid unsaturation and neutral lipid on the maturation of high density lipoproteins, the discoidal complexes of apoA-I, phosphatidylcholine and cholesteryl ester (CE) were prepared. Saturated dipalmitoylphosphatidylcholine (DPPC) and unsaturated palmitoyllinoleoylphosphatidylcholine (PLPC), palmitoyloleoylphosphatidylcholine (POPC), and fluorescent probe cholesteryl 1-pyrenedecanoate (CPD) that forms in a diffusion- and concentration-dependent manner short-lived dimer of unexcited and excited molecules (excimer) were used. The apoA-I/DPPC/CPD complexes were heterogeneous by size, composition and probe location. CPD molecules incorporated more efficiently into larger complexes and accumulated in a central part of the discs. The apoA-I/POPC(PLPC)/CPD were also heterogeneous, however, probe molecules distributed preferentially into smaller complexes and accumulated at disc periphery. The kinetics of CPD transfer by recombinant cholesteryl ester transfer protein (CETP) to human plasma LDL is well described by two-exponential decay, the fast component with a shorter transfer time being more populated in PLPC compared to DPPC complexes. The presence of CE molecules in discoidal HDL results in particle heterogeneity. ApoA-I influences the CETP activity modulating the properties of apolipoprotein–phospholipid interface. This may include CE molecules accumulation in the boundary lipid in unsaturated phosphatidylcholine and cluster formation in the bulk bilayer in saturated phosphatidylcholine.     

液相色谱-串联质谱法检测人血浆及脂蛋白唾液酸的含量

建立了液相色谱-串联质谱(LC-MS/MS)检测人血浆脂蛋白唾液酸的方法,并比较了糖尿病患者与健康人血浆脂蛋白唾液酸的差异。采用pH=2的醋酸水解唾液酸,高速离心后采用优化的条件进行LC-MS/MS分析。结果表明:在负离子模式下,唾液酸的检出限和定量限分别为7.4和24.5 pg。唾液酸在2.5~80 ng/mL范围内呈良好的线性关系,相关系数R²大于0.998。糖尿病患者(平均年龄51.6岁)和健康人(平均年龄50.7岁)血浆中唾液酸的含量分别为(548.3±88.9)和(415.3±55.5)mg/L;两组实验人群中极低密度脂蛋白、低密度脂蛋白和高密度脂蛋白唾液酸的含量分别为(4.91±0.19)、(6.95±0.28)、(3.61±0.22)μg/mg和(2.90±0.27)、(7.03±0.04)、(2.40±0.09)μg/mg。糖尿病患者极低密度脂蛋白和高密度脂蛋白唾液酸含量显著高于同龄健康人水平(P<0.01)。该法可快速检测血浆中脂蛋白唾液酸含量,省时省力。     

Nonaqueous capillary electrophoresis for analysis of the ethanol consumption biomarker phosphatidylethanol

Nonaqueous CE (NACE) methodology was developed for the separation and determination of phosphatidylethanol (Peth), a new biomarker of ethanol intake. Peth is an abnormal phospholipid formed in cell membranes only in the presence of ethanol, via the transphosphatidylation reaction of phospholipase D. The NACE separation medium consisted of 80 mM ammonium acetate in 50% ACN, 33% 2-propanol, 12% hexane and 5% methanol. A stacking effect was obtained by reducing the concentration of ammonium acetate in the separation medium for all injected samples. The LOD was estimated to 1 microM (5.6 fmol) of Peth with conventional UV detection, equalling 0.4 micromol/L blood. Peth was successfully determined in extracts of human blood samples. Separation of Peth from other blood lipids in the lipid extract sample was performed in 5 min. The method facilitates smaller sample volumes and performs about ten times faster compared to earlier chromatographical methods.     

Monitoring of Polymerized Triglycerides in Deep-Frying Oil by On-Line GPC-FTIR Spectrometry Using the Science Based Calibration Multivariate Approach

On-line gel permeation chromatography Fourier transform infrared (GPC-FTIR) is proposed as detection technique for the determination of the total polymer triacylglyceride (PTG) content in olive oil as an alternative to other conventional detectors such as the evaporative light scattering detector (ELSD) or the refraction index detector (RID). FTIR detection allowed confirmation additionally to quantification of the analytes via their characteristic infrared spectra. For the extraction of chromatograms, a multivariate method called science based calibration (SBC) was used. The SBC method employed a reference spectrum of triacylglycerides (TG) extracted from the injection of a fresh olive oil sample and data obtained from a blank injection as ‘noise’ matrix, to estimate a regression vector subsequently used to predict the relative concentration of the analyte during the GPC run. Results found evidenced that the use of SBC improved the selectivity, sensitivity and repeatability of the GPC-FTIR measurements as compared to the ‘classical’ approach based on the measurement of the spectral area in a defined spectral range. The developed method provided a limit of detection for PTGs of 0.19 mg mL^?1, which corresponds to a 0.65% w/w for an oil sample mass of 300 mg.     

Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry

The separation and characterization of human apolipoproteins and their isoforms was investigated using capillary electrophoresis (CE) in combination with mass spectrometry (MS). The focus of these analyses was the major protein constituents of plasma high-density lipoproteins, apolipoprotein A-I and A-II. Using aqueous buffers in CE, no separation between apolipoprotein A-I and A-II was observed. With the addition of 10-20% acetonitrile, however, the two species could be separated. Furthermore, multiple peaks for each of the apolipoprotein species were observed under these CE conditions. In order to identify and characterize the components, these separations were then coupled with online mass spectrometric detection (CE-MS). Our CE-MS results suggest that the multiple components observed in the acetonitrile-containing CE separation appear to be oxidized forms of the proteins in addition to native forms of the apolipoprotein A-I and A-II. These data are in agreement with previous reports that the methionine residues of the high-density lipoproteins (HDLs) are sensitive to oxidation, which in turn, alters their lipid binding characteristics and secondary structure. In addition to oxidized forms of the proteins, apolipoprotein A-II contained additional components, which varied in mass by 128 Da. The structural differences between these components were determined by proteolytic digestion and tandem MS. Using these techniques, we determined that these components were due to truncation of the C-terminal glutamine amino acid residue on apolipoprotein A-II. These results demonstrate that CE in combination with MS is a promising technique for screening and characterizing isomers of plasma apolipoproteins.     

Distribution of protease inhibitors in lipid emulsions: gabexate mesilate and camostat mesilate

Gabexate mesilate (GM) and camostat mesilate (CM) are protease inhibitors used for the treatment of pancreatitis, and have been reported to show anticancer effects in vivo. Lipid emulsions (20% fractionated soybean oil) were investigated in terms of physicochemical interaction between the drugs and lipid emulsions as a possible drug carrier. The result showed that the drugs did not distribute in the oil phase but were adsorbed at the phospholipid interface of oil droplets. With increasing concentration of the drugs, the adsorption amount at the interface rose steeply to around 2.2x10(-11) mol/cm2 for GM and 1.2x10(-11) mol/cm2 for CM, respectively, followed by further adsorption deviated from the Langmuir adsorption manner after the inflection. To interpret this two-stage adsorption of the drugs, surface potential and fluorescence changes were examined in addition to thermodynamics for their interaction with the interfacial lipid layer. The primary adsorption was exothermic and was due to electrostatic interaction and van der Waals interaction between drug molecules and phospholipid molecules. Both acidic and neutral phospholipids in the lipid were involved in the adsorption of GM, while acidic phospholipids were mainly involved in the adsorption of CM. On the other hand, the secondary adsorption was endothermic and was entropy-driven most probably due to hydrophobic interaction for GM and CM in common, including peripheral penetration of drug molecules into the interfacial lipid layer.