聚二甲基硅氧烷(PDMS)/玻璃微流控芯片电泳快速分离血清高密度脂蛋白亚类及临床应用研究

经梯度密度超速离心,高密度脂蛋白(HDL)分为HDL2和HDL3两亚型。HDL2抑制低密度脂蛋白(LDL)氧化功能受损是冠心病(CHD)发生发展的关键因素。因此,通过对HDL亚类进行分离,从而达到预测和诊断CHD的目的。本研究建立了用PDMS/玻璃微流控芯片快速电泳分离HDL亚类的方法。选择N-十二烷基-β-D-麦芽糖苷(DDM)、十二烷基硫酸钠(SDS)和羟丙基纤维素(HPC)共同修饰脂蛋白和泳道表面。在以含0.3 mmol/L SDS的50 mmol/L 3-(N-吗啉代)丙磺酸(MOPS)(pH 8.0)为样品缓冲液,含0.6%HPC的50 mmol/L MOPS(pH 8.0)为分离缓冲液,分离电压为260 V/cm的优化条件下,HDL2和HDL3在4 min内得到基线分离,二者的出峰时间和峰面积的相对标准差(RSD)分别是2.0%和2.7%,2.0%和2.9%,具有较好的重复性。临床标本研究发现,正常人血清标本可分离出HDL2和HDL3双峰,而CHD患者的HDL2峰面积显著减小,甚至消失。PDMS/玻璃微流控芯片分离HDL亚类是一种简单、快速、高效的用于分析CHD危险因子的方法。     

Application of poly(dimethylsiloxane)/glass microchip for fast electrophoretic separation of serum small,dense low-density lipoprotein

Due to the mounting evidence of altered low-density lipoprotein (LDL) size in several disease states, there has been an increasing interest in developing new analytical methods for small, dense low-density lipoprotein (sdLDL) for diagnosis. The present report demonstrates that sdLDL analysis can be performed in a poly(dimethylsiloxane) (PDMS/glass) microchannel. n-Dodecyl β-d-maltoside (DDM) was utilized to alter channel surface to make it become hydrophilic and nonionic, thus reducing the interaction between the protein and the surface. Moreover, hydroxypropylcellulose (HPC) was added into the running buffer to suppress the adsorption of analytes and also to serve as a sieving matrix. Under optimal conditions, two baseline separations of lipoproteins including high-density lipoprotein (HDL), sdLDL, and lLDL were achieved with different selectivity. LDL particles shown on the electropherogram were also identified by several procedures. This method affords high separation speed and high reproducibility. The intraassay and interassay RSDs of lipoprotein migration times were in the range of 2.01–2.45%. The variation of serum sdLDL of a patient between prior treatment and post-treatment was assessed by this method. This system has the potential for rapid and sensitive detection of different LDL forms, and thus will be applicable to clinical diagnosis.     

微流控芯片电泳分离血清中小而密低密度脂蛋白的研究

应用微流控芯片电泳,以40 mmol/L Tricine(pH9.8)作为电泳缓冲体系,十二烷基硫酸钠(SDS)作为添加剂(0.1 mmol/L SDS样品溶液,0.02 mmol/L SDS分离缓冲液),分离血清小而密低密度脂蛋白(sdLDL)。研究荧光染料硝基苯并噁二唑-C6-酰基鞘胺醇(NBD C6-ceramide)与脂蛋白结合的特异性、饱和性以及血清保存和检测时间对脂蛋白电泳行为的影响;探讨SDS有效降低蛋白吸附,提高血清脂蛋白分辨率的作用。冠心病(CHD)组sdLDL检出率(75%)显著高于对照组(6%,P<0.01)。该法具有简易、快速、高效等优点,可望成为CHD危险性评估的常规分析手段。     

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.     

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.

     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol.

Human VLDL, LDL and HDL (very-low-, low-, and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

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.     

微流控芯片电泳快速分离脂蛋白

描述了一种芯片电泳快速分离脂蛋白的方法. 利用自制的微流控芯片及激光诱导荧光技术电泳分离经硝基苯并噁二唑-C₆-酰基鞘胺醇预染的脂蛋白标本, 在40 mmol/L tricine缓冲液(pH 9.4)中加入40 mmol/L甲基葡胺, 在500 V电压下40 s进样, 在2000 V 电压下2 min内完成分离, 可出现低密度脂蛋白(LDL)与高密度脂蛋白(HDL)两条脂蛋白区带, 5次重复性试验其出峰时间变异系数(CV)为2.6%. 本法为高血脂患者提供了一种快速、简便、灵敏、重复性好的诊断方法.     

Ellipsometry Studies of Lipoprotein Adsorption

The adsorption of a number of lipoproteins, i.e., low-density lipoprotein (LDL), oxidized LDL (oxLDL), high-density lipoprotein (HDL), and lipoprotein (a), at silica and methylated silica as well as at the latter surface modified through adsorption of proteoheparan sulfate, was investigated with in situ ellipsometry at close to physiological conditions. It was found that LDL, oxLDL, HDL, and lipoprotein (a) all adsorbed more extensively at silica than at methylated silica. Upon exposure of the methylated silica surface to proteoheparan sulfate, this proteoglycan adsorbs through its hydrophobic moiety, thereby forming a layer similar to that in the biological system, with the polysaccharide chains forming brushes oriented toward the aqueous solution. Analogous to the biological system, both lipoprotein (a) and LDL were found to deposit at such surfaces, the latter particularly in the simultaneous presence of Ca(2+). After HDL pre-exposure, however, no LDL deposition was observed, even at high LDL and Ca(2+) concentrations. These findings correlate well with those obtained from clinical investigations on risk factors for atherosclerosis. Copyright 2000 Academic Press.     

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.     

Characterization of modified low density lipoprotein subfractions by capillary isotachophoresis

Oxidative modification of low density lipoproteins (LDLs) is an important pathogenetic factor in atherosclerosis. The various steps in oxidative modifications of LDL can be monitored using different methodologies with varying degrees of complexity. In this study, we propose capillary isotachophoresis (CITP) as a suitable tool to detect and measure the degree of oxidation of LDL. LDL was isolated from pooled plasma of healthy volunteers by sequential ultracentrifugation, and oxidation was performed in vitro as well as in cell culture experiments. Native LDL and oxidatively modified LDL were characterized by apo B-100 fluorescence and conjugated diene formation. Samples were separated by CITP combined with sudan black B staining. To underline the inherent advantages of this approach, CITP was compared with classical lipoprotein electrophoresis using agarose gel. We demonstrate the CITP method to be highly sensitive, as changes in peak area of the separated LDL subfractions were detected after only 2 h of oxidation. The leading LDL peaks increased, while the terminating LDL peaks decreased in parallel throughout the duration of oxidation. The LDL samples, oxidized for 4-24 h, also exhibited an increased migration velocity of the fractions. In summary, we present the first study investigating LDL-subfractions separated by CITP and the alterations of these LDL-subfractions after gradual in vitro oxidation and after oxidative modification by monocyte-derived macrophages and vascular smooth muscle cells.     

Antibody–Ferrocene Conjugates as a Platform for Electro-Chemical Detection of Low-Density Lipoprotein

Low-density lipoprotein (LDL) is a cardiac biomarker identified in the pathology of cardiovascular disease (CVD). Typically, the level of LDL is calculated using the Friedewald relationship based on measured values of total cholesterol, high-density lipoproteins (HDL), and triglycerides. Unfortunately, this approach leads to some errors in calculation. Therefore, direct methods that can be used for fast and accurate detection of LDL are needed. The purpose of this study was to develop an electrochemical platform for the detection of LDL based on an antibody–ferrocene conjugate. An anti-apolipoprotein B-100 antibody labeled with ferrocene was covalently immobilized on the layer of 4-aminothiophenol (4-ATP) on the surface of gold electrodes. Upon interaction between LDL and the antibody–ferrocene conjugate, a decrease in the ferrocene redox signal registered by square wave voltammetry was observed, which depends linearly on the concentration from 0.01 ng/mL to 1.0 ng/mL. The obtained limit of detection was equal to 0.53 ng/mL. Moreover, the satisfied selectivity toward human serum albumin (HSA), HDL, and malondialdehyde-modified low-density lipoprotein (MDA-LDL) was observed. In addition, the acceptable recovery rates of LDL in human serum samples indicate the possible application of immunosensors presented in clinical diagnostics.     

Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography–tandem mass spectrometry

This study describes a coupled analytical method to carry out the systematic profiling of phospholipids (PLs) in high-density lipoproteins (HDL) and low-density lipoproteins (LDL) from human blood plasma. HDL and LDL of healthy human plasma samples were separated by size and collected on a semi-preparative scale using multiplexed hollow fiber flow field-flow fractionation (MxHF5). Phospholipid mixtures contained in the resulting HDL and LDL fractions were analyzed by shotgun nanoflow liquid chromatography–tandem mass spectrometry (nLC–ESI-MS–MS). We utilized a dual scan method for the separation and simultaneous characterization of complicated PL mixtures by nLC–ESI-MS–MS, such that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules were detected in positive ion mode in a first LC run. In a second LC run, phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) were detected in negative ion mode. In this study, a total of 56 PLs from HDL and 52 PLs from LDL particles were characterized by their molecular structures from data dependent collision-induced dissociation (CID) experiments, and their relative abundances were compared.     

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     

Electrophoretic behavior and partial characterization of disease-associated serum forms of gamma-glutamyltransferase

We have recently devised an improved procedure for the rapid electrophoretic separation of multiple forms of serum gamma-glutamyltransferase (GGT). This procedure is based on the separation on cellulose acetate strips, usually employed for lipoprotein electrophoresis, followed by visualization with a fluorescent reagent. The method is highly sensitive and the fractions are more clearly resolved than with other procedures. Reference intervals have been evaluated in the sera from 142 healthy subjects and the patterns (two GGT forms comigrating with alpha 1 and alpha 2-globulin) are reproducible. In 150 sera from patients with various hepatobiliary diseases (including neoplasias), acute pancreatitis and non liver-involving neoplasias, we observed some disease-specific GGT forms: an albumin comigrating enzyme (Alb-GGT) specific of liver neoplasia; a gamma-globulin comigrating GGT (gamma-GGT) and a nonmigrating isoform (dep-GGT) both specifically associated to extrahepatic jaundice. Multiple lipoprotein fraction precipitation showed that beta-, gamma- and dep-GGT are complexes between GGT and low density lipoprotein and very low density lipoproteins (LDL + VLDL), and that some of the alpha 1-GGT from cirrhotic patients is a complex between GGT and high density lipoprotein (HDL). GGT fractions from normal subjects and Alb-GGT from patients with liver neoplasia do not appear to be complexed with lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS–PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI–MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI–MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.     

Analysis of Fatty Acid, and Lipoprotein, Metabolism by GC and HPLC: Effect of Low-Density Lipoprotein Apheresis

 Blood samples from 5 hyperlipidemic patients on chronic treatment with low-density lipoprotein (LDL) – apheresis were analysed for lipids and fatty acids in serum, lipoprotein fractions and erythrocyte membrane by capillary gas chromatography (GC), reversed-phase high-performance liquid chromatography (LC), spectrofluorometry and spectrophotometry. LDL-apheresis has been associated with significant changes of fatty acids metabolism in relation to triglyceride-rich lipoproteins. Oleic acid may exert its hypotriglyceridemic effect via VLDL, IDL, LDL and HDL fractions. Polyunsaturated fatty acids, associated with triglyceride metabolism via IDL or VLDL, are linoleic, gamma-linolenic and docosahexaenoic fatty acids. Received November 25, 1999. Revision September 5, 2000.     

A Microchip-Based Method for Rapid Separation of Subclasses of High-Density Lipoprotein

The subclasses of high-density lipoprotein (HDL) labeled with NBD C₆-ceramide have been rapidly separated by microfluidic chip electrophoresis and detected by use of laser-induced fluorescence. The subclasses HDL₂ and HDL₃ were separated in 4 min. Results showed that the HDL₂/HDL₃ ratio for patients with CHD was much lower than that for healthy subjects. This method could meet the demand for clinical examination of HDL subclasses.

     

A novel method for serum lipoprotein profiling using high performance capillary isotachophoresis

A new capillary isotachophoresis (cITP) method for lipoprotein profiling with superior lipoprotein coverage compared to previous methods has been developed, resolving twice as many lipoprotein species (18 peaks/fractions) in serum or plasma in less than 9.5 min. For this, a novel mixture of 24 spacers, including amino acids, dipeptides and sulfonic acids, was developed and fine-tuned, using predictive software (PeakMaster) and testing of spiked serum samples. Lipoprotein peaks were identified by serum-spiking with reference lipoproteins. Compatibility with common lipophilic stains for selective lipoprotein detection with either UV/Vis or laser-induced fluorescence was demonstrated. A special new capillary with a neutral coating (combining water-compatible OV1701-OH deactivation and methylation) was used for the first time for electrodriven separations, allowing very stable separations in a pH 8.8–9.4 gradient system, being functional for more than 100 injections. Excellent reproducibility was achieved, with coefficients of variation lower than 2.6% for absolute migration times. Comparison was performed with human plasma samples analyzed by NMR, leading to similar results with cITP after multivariate statistics, regarding group-clustering and lipoprotein species correlation. The new cITP method was applied to the analysis of serum samples from a LDL receptor knock-out mice model fed either a normal diet or a western-type diet. Differences in the lipoprotein levels and in the sublipoprotein types were detected, showing a shift to more atherogenic particles due to the high cholesterol diet. In summary, this novel method will allow more detailed and informative profiling of lipoprotein particle subtypes for cardiovascular disease research.     

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.