Enantioselective binding analysis of verapamil to plasma lipoproteins by capillary electrophoresis-frontal analysis

Capillary electrophoresis coupled with frontal analysis was applied to the study of enantioselective binding of verapamil (VER) to plasma lipoproteins. The drug-lipoprotein mixed solution, which had been in the binding equilibrium, was hydrodynamically introduced into a non-coated fused-silica capillary. Since VER is positively charged in the neutral run buffer (pH 7.4), the unbound VER enantiomers migrated toward the cathodic end much faster than negatively charged lipoproteins and their bound forms. Once unbound VER migrated apart from lipoprotein, the bound VER was quickly released from the protein to maintain the binding equilibrium. Thus, VER migrated as a zone through the capillary and gave a trapezoidal peak with a plateau region on the electropherogram. The VER concentration in this plateau region was equal to the unbound VER concentration in the initial sample solution. It was found that the bindings of VER to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL were not site-specific and not enantioselective. Partition-like binding to lipid part of these lipoproteins seemed to be dominant. The total binding affinities of LDL to VER were about seven-times stronger than those of HDL, and the oxidation of LDL by copper ion enhanced the binding affinities significantly.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixture of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10(4) and 10(5). HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Low-density lipoprotein analysis in microchip capillary electrophoresis systems

Due to the mounting evidence for altered lipoprotein and cholesterol-lipoprotein content in several disease states, there has been an increasing interest in analytical methods for lipoprotein profiling for diagnosis. The separation of low- and high-density lipoproteins (LDL and HDL, respectively) has been recently demonstrated using a microchip capillary electrophoresis (CE) system [1]. In contrast to this previous study, the present report demonstrates that LDL analysis can be performed in an uncoated glass microchannel. Moreover, by adding sodium dodecyl sulfate (SDS) to the sample at a concentration well below the critical micellar concentration prior to injection, the LDL peak undergoes a focusing effect and exhibits an apparent efficiency of 2.2 x 10(7) plates/m. Laser light scattering experiments demonstrate that the low concentration of SDS used does not significantly alter lipoprotein particle size distribution within the time course that the analysis is performed. It is thus hypothesized that SDS nondisruptively coats LDL particles. The peak sharpening effect, observed only when SDS is added solely to the sample, is probably due to a mobility gradient created between the sample and the running buffer. The chip-based method demonstrated here has the potential for rapid analysis and sensitive detection of different LDL forms of clinical relevance.     

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.     

Photochemistry of Bacteriochlorophylls in Human Blood Plasma: 1. Pigment Stability and Light-induced Modifications of Lipoproteins

Transmetalated derivatives of bacteriochlorophyll are promising sensitizers in photodynamic therapy. Protocols using short delay times between injection and irradiation cause interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the photochemistry of Zn- and Pd-bacteriopheophorbide (WST09), and of the highly polar taurinated Pd-derivative, WST11, in isolated fractions of human blood plasma. The stability of all pigments is increased in blood plasma, compared with monomeric solutions. Pd-bacteriopheophorbide is much more stable than the other two derivatives. It also has a higher capacity for inducing reactive oxygen species, yet the consumption of oxygen is comparable. There is furthermore evidence for photobleaching under anoxic conditions. The generation of hydroperoxides (ROOH) is faster with Pd- than with Zn-complexes; the formation of endoperoxides (ROOR′), measured as thiobarbituric acid reactive substances, is comparable with the two central metals. Formation of both ROOH and ROOR′ is increased in low-density lipoproteins (LDL) compared with high-density lipoproteins (HDL), which is probably related to the higher concentration of target molecules in the former. In HDL, extensive cross-linking is induced among the apolipoproteins; judged from the electrophoretic mobility of LDL and HDL particles, there is also a gross structural change. Photosensitized cross-linking is much less pronounced with high-density proteins.     

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。     

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.     

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.     

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.     

Reaction of fluorogenic reagents with proteins II: capillary electrophoresis and laser-induced fluorescence properties of proteins labeled with Chromeo P465

The fluorogenic reagent Chromeo P465 is considered for the analysis of proteins by capillary electrophoresis with laser-induced fluorescence detection. The reagent was first used to label alpha-lactalbumin; the product was analyzed by capillary zone electrophoresis in a sub-micellar sodium dodecyl sulfate (SDS) buffer. The product generated a set of equally spaced but poorly resolved peaks that formed a broad envelope with a net mobility of 4 x 10(-4)cm(2) V(-1) s(-1). The components of the envelope were presumably protein that had reacted with different numbers of labels. The mobility of these components decreased by roughly 1% with the addition of each label. The signal increased linearly from 1.0 nM to 100 nM alpha-lactalbumin (r(2)=0.99), with a 3sigma detection limit of 70 pM. We then considered the separation of a mixture of ovalbumin, alpha-chymotrypsinogen A, and alpha-lactalbumin labeled with Chromeo P465; unfortunately, baseline resolution was not achieved with a borax/SDS buffer. Better resolution was achieved with N-cyclohexyl-2-aminoethanesulfonic acid/Tris/SDS/dextran capillary sieving electrophoresis; however, dye interactions with this buffer system produced a less than ideal blank.     

Erythrocyte spectrin alteration induced by low-density lipoprotein.

Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylation-dephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.     

Determination of synthetic food dyes by capillary electrophoresis

A method for the determination of synthetic tar dyes used as food additives using capillary electrophoresis with photodiode-array detection was investigated. The dyes Erythrosine (R-3), Phloxine (R-104), Rose Bengal (R-105), Acid Red (R-106), Amaranth (R-2), New Coccine (R-102) and Allura Red AC (R-40) were separated on a capillary column (50 cm × 75 μm I.D.) and identified from the absorbance spectra of each peak. The electrophoresis buffer used was a mixture of 25 mM sodium phosphate buffer and 25 mM sodium borate buffer (1:1) (pH 8.0) containing 10 mM sodium dodecyl sulfate (SDS). Substitution of β-cyclodextrin for SDS in the electrolyte buffer was effective for the separation of R-2 and R-102. This modified method could be employed as an additional assay method for these two dyes.     

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.

     

Buffers to suppress sodium dodecyl sulfate adsorption to polyethylene oxide for protein separation on capillary polymer electrophoresis

Although polyethylene oxide (PEO) offers several advantages as a sieving polymer in SDS capillary polymer electrophoresis (SDS-CPE), solution properties of PEO cause deterioration in the electrophoresis because PEO in solution aggregates itself, degrades into smaller pieces, and forms polymer-micelle complexes with SDS. We examined protein separation on SDS-CPE with PEO as a sieving matrix in four individual buffer solutions: Tris-CHES, Tris-Gly, Tris-Tricine, and Tris-HCl buffers. The solution properties of PEO as a sieving matrix in those buffers were examined by dynamic light scattering (DLS) and by surface tension. Preferential SDS adsorption onto PEO disturbed protein-SDS complexation and impaired the protein separation efficiency. Substantial adsorption of SDS to PEO was particularly observed in Tris-Gly buffer. The Tris-CHES buffer prevented SDS from adsorbing onto the PEO. Only Tris-CHES buffer achieved separation of six proteins. This study demonstrated efficient protein separation on SDS-CPE with PEO.     

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.     

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.     

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.     

UVB-INDUCED PHOTOPEROXIDATION OF LIPIDS OF HUMAN LOW and HIGH DENSITY LIPOPROTEINS. A POSSIBLE ROLE OF TRYPTOPHAN RESIDUES

Ultraviolet radiation of the UVB region readily destroy tryptophan (Trp) residues of low (LDL) and high (HDL) density lipoproteins. The photooxidation of tryptophan residues is accompanied by the peroxidation of low and high density lipoproteins unsaturated fatty acids, as measured by the thiobarbituric acid assay. Moreover, low and high density lipoproteins are natural carriers of vitamin E and carotenoids. These two antioxidants are also rapidly bleached by UVB. The UVA radiation promotes neither tryptophan residue destruction nor lipid photoperoxidation. The redox cycling Cu2+ ions considerably increase lipid photoperoxidation. The synergistic action of photo and auto (Cu2(+)-induced) peroxidation induces marked post-irradiation modifications of apolipoproteins as illustrated by the degradation of most tryptophan residues after overnight incubation in the dark of pre-irradiated samples.     

Microchip-based small, dense low-density lipoproteins assay for coronary heart disease risk assessment

Small, dense low-density lipoprotein (sdLDL) has been accepted as an emerging cardiovascular risk factor, and there has been an increasing interest in analytical methods for sdLDL profiling for diagnosis. Serum sdLDL may be measured by different laboratory techniques, but all these methods are laborious, time-consuming, and costly. Recently, we have demonstrated that a low-temperature bonding of quartz microfluidic chips for serum lipoproteins analysis (Zhuang, G., Jin, Q., Liu, J., Cong, H. et al., Biomed. Microdevices 2006, 8, 255-261). In contrast to this previous study, we chose SDS as anionic surfactant to modify both lipoproteins and the channel surface to minimize lipoprotein adsorption and improve the resolution of lipoprotein separation. Two major LDL subclass patterns including large, buoyant LDL (lLDL), sdLDL, and high-density lipoprotein (HDL) were effectively separated with high reproducibility. RSD values of the migration time (min) and peak areas of standard LDL and HDL were 6.28, 4.02, 5.02, and 2.5%, respectively. Serum lipoproteins of 15 healthy subjects and 15 patients with coronary heart disease (CHD) were separated by microchip CE. No peaks of sdLDL were detected in serum samples of healthy subjects while sdLDL fractional peaks were observed in patients' entire serum samples. These results suggested that the microchip-based sdLDLs assay was a simple, rapid, and highly efficient technique and significantly improved the analysis of CHD risk factors.