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.     

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.

     

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.     

Comparative analysis of foetal calf and human low density lipoprotein: relevance for pharmacodynamics of photosensitizers

The effects of differences in lipoprotein content on the distribution of the novel hydrophobic photosensitizer n-butyl-3-[18-(2-butylcarbamoyl-ethyl)-3,7,12,17-tetramethyl-18,13-divinyl-22,24-dihydro-porphin-2-yl]propionamide (PP-N-3) and haematoporphyrin ester (HpE), a relatively hydrophilic photosensitizer, in human (HS) and foetal calf sera (FCS), were investigated. The binding characteristics of human and foetal calf low-density lipoprotein (LDL) were characterised using a human fibroblast line (Vag 12). The uptake into cells of HpE and PP-N-3 was also examined. A comparison of the lipoprotein content, composition and receptor-binding characteristics of foetal calf and human serum was also carried out. LDL content was measured directly using sequential ultracentrifugation to isolate LDL. In our study, we found haematoporphyrin ester to bind to human very low-density lipoprotein (VLDL), LDL and high-density lipoprotein (HDL) in the ratio 2:31:65. In the case of PP-N-3 this ratio was 56:10:33. As VLDL was not detected in foetal calf serum, only binding to LDL and HDL was observed. Using the sequential ultracentrifugation technique, foetal calf serum was found to contain LDL which in turn did bind to human LDL receptors. The uptake of PP-N-3 and HpE in the presence of low density lipoprotein from foetal calf serum (FC-LDL) was not significantly different to values observed in the presence of human serum low density lipoprotein (HS-LDL).     

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。     

Adsorption/Desorption of Low-density Lipoprotein on a Heparinized Surface of Gold Sensors

Heparin has been considered to be a potentially useful ligand for low-density lipoprotein(LDL)detection and analysis in a clinical context.In order to construct an affinity surface for preferential ads...     

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.     

Fabrication of a label-free electrochemical immunosensor of low-density lipoprotein

The silver chloride@polyaniline (PANI) core-shell nanocomposites (AgCl@PANI) combined with Au nanoparticles (AuNPs) were used to prepare the AuNPs-AgCl@PANI hybrid material. A novel sensitive label-free low-density lipoprotein (LDL) electrochemical biosensor was fabricated by adsorption of antibody to apolipoprotein B-100 (aopB-100) on an AuNPs-AgCl@PANI-modified glassy carbon (GC) electrode. The hybrid material could provide surface for high antibody loading due to its large surface-to-volume ratio. Since each LDL has an apoB-100 on its phospholipids coat, they could be bonded to the electrode surface through the specific antibody-antigen reaction. Electrochemical impedance spectroscopy (EIS) was used to characterize the recognition of LDL. The negative charges carried by LDL phospholipids coat would block the electron transfer of the [Fe(CN)6]3-/4- redox couple severely. In addition, the conductivity of LDL is very poor, so small amounts of LDL on the electrode could result in great change in the electron-transfer resistance (Ret). The biosensor exhibited a highly sensitive response to LDL with a detection limit of 0.34 pg/mL, and some factors that would affect the performance of the biosensor were studied, such as incubation time and temperature.     

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.     

聚丙烯微孔膜表面肝素化及其对低密度脂蛋白的吸附特性

为了赋予聚丙烯微孔膜(PPMM)选择性吸附低密度脂蛋白(LDL)的能力,发展了一种有效的PPMM表面共价固定肝素的方法.基于紫外引发丙烯酸的接枝聚合,通过碳二亚胺活化,以乙二胺为间隔臂,将肝素共价固定于PPMM表面,获得表面肝素化的PPMM.ATR-FTIR和XPS分析确证了修饰过程中膜表面基团及化学成分的变化.采用静...     

High level of low-density lipoprotein receptors enhance hypericin uptake by U-87 MG cells in the presence of LDL

The dependence of the uptake of hypericin (Hyp) by human glioma U-87 MG cells on the level of expression of low-density lipoprotein (LDL) receptors has been studied in this work. A special role of the LDL receptor-pathway for Hyp delivery to U-87 MG cells in the presence of LDL was revealed by the substantial increase of Hyp uptake in the situation, when the number of LDL receptors on the cell surface was elevated. Moreover, the colocalization experiments showed the lysosomal localization of Hyp following the uptake and that the concentration of Hyp in these organelles was enhanced in the cells with elevated number of LDL receptors when the incubation medium contained LDL. Both these findings suggest that LDL and LDL receptor-pathway play an important role in the delivery and accumulation of Hyp into the cells.     

Cyclic diarylheptanoids inhibit cell mediated low-density lipoprotein oxidation

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC(50) values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than 5 times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. Compound (1) had no effect on the anti-atherogenesis in low-density lipoprotein receptor-deficient mice.     

一种新型低密度脂蛋白吸附剂的制备

研究表明,人体血浆中低密度脂蛋白(LDL)的含量过高会引起动脉硬化,进而诱发各种心脑血管疾病^[1].近年来,采用血液净化吸附剂去除LDL受到了广泛的关注^[2～10].目前临床上使用的吸附剂主要有固载LDL抗体的琼脂糖吸附剂及固载肝素或磺化葡聚糖的纤维素吸附剂,它们虽有较好的吸附效果,但由于存在配基昂贵、吸附剂成本高及机械强度差等问题,这些吸附剂的应用受到了很大的限制.本文以具有良好机械强度和血液相容性的聚乙烯醇(PVA)大孔珠状树脂为载体,以廉价易得的吲哚-3-乙酸(IAA)为配基,分别以不同长度的多胺为悬臂,制备了一种新型LDL吸附剂,并初步考察了其对LDL的吸附性能.     

Recent progress in the detection and treatment of atherosclerosis by nanoparticles

Cardiovascular diseases (CVDs) have high mortality and morbidity in the US and presently rank as one of the leading causes of death. Atherosclerosis (AS) acts as one of the CVDs, playing an important role in mortality because of many lethal complications. The common cause of AS is that low-density lipoprotein (LDL) in the blood circulation enters the intima through endothelial cells that have been broken for various reasons. Under the action of inflammatory factors secreted by damaged endothelial cells, monocytes also enter the inner membrane and differentiate into macrophages. Macrophages engulf the oxidized LDL and become foam cells which eventually become apoptotic. However, recent studies have shown that LDL entry into the intima, an important step in AS, may be associated with endothelial cells actively inhaling LDL through the receptor. Nanotechnology is a promising technology that can be applied in the noninvasive imaging and therapy of AS. Nanoparticles (NPs) have the ability to passively target AS because of their inherent small diameter. They can also be loaded with chemicals for targeting lesions, contrast agents for imaging, and drugs for treatment to achieve accurate diagnosis and treatment of AS. This review consequently highlights the recent progress in the detection and treatment of AS by NPs.     

Cyclic diarylheptanoids inhibit cell-mediated low-density lipoprotein oxidation

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC50 values of 2.9 and 1.7 microM, respectively, and they also inhibited cell-mediated LDL oxidation more than five times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 microM. 1 had no effect on the anti-atherogenesis in low-density lipoprotein receptor- deficient mice.     

Rapid method for measuring plasma low-density lipoprotein turnover using high-performance gel exclusion chromatography

High-performance gel exclusion chromatography using flow-through radioactivity monitoring was employed for rapid measurement of low-density lipoprotein (LDL) turnover. Iodinated LDL was injected into five fasting rabbits. Serial blood samples were obtained and small aliquots (0.1-0.2 ml) were injected into the chromatographic system using 0.2 M Tris-acetate buffer (pH 7.0). Radioactivity in lipoproteins was measured by an on-line gamma counter (Flo-One) attached to the high-performance liquid chromatograph and in a regular gamma counter after collecting the fractions. Sequential ultracentrifugation was also used to separate lipoproteins, and the radioactivity in each fraction was measured. The Flo-One method was faster; however, the efficiency of Flo-One varied with the amount of radioactivity and therefore it was necessary to inject the same amount of radioactivity to get comparable values.     

Relationship between Apolipoprotein E Genotype and Lipoprotein Profile in Patients with Coronary Heart Disease

(1) Background: Apolipoprotein E(ApoE) plays a critical role in lipid transport. The specific allele of APOE being expressed is associated with the development of coronary heart disease (CHD), however the specific mechanisms by which ApoE drives disease are unclear. In this study, we investigated the relationship between APOE allele, lipoprotein metabolome, and CHD severity to provide evidence for the efficacy of clinical cholesterol-lowering therapy; (2) Methods: Blood samples were collected from 360 patients with CHD that were actively being treated with statins. The lipoprotein profile, including particle numbers, particle size, and lipoprotein composition concentrates, was measured by nuclear magnetic resonance (NMR) spectroscopy. The severity of CHD was determined by quantifying coronary angiography results using the Gensini scoring system; (3) Results: We found there was no significant difference in low-density lipoprotein cholesterol (LDL-C) levels among ε2+ (ε2 allele carriers, consisting of ε2/ε2 and ε2/ε3 genotypes), ε3 (consisting of ε3/ε3 and ε2/ε4 genotypes), and ε4+ (ε4 allele carriers, consisting of ε3/ε4 and ε4/ε4 genotypes) participants receiving statin treatment. Compared with the ε3 group, patients with the ε2+ genotype showed lower concentrations of total low-density lipoprotein (LDL), small-LDL, and middle-LDL particles, as well as a larger LDL size, higher very low-density lipoprotein (VLDL) composition concentrates, and higher intermediate density lipoprotein (IDL) composition concentrates. The ε4+ group showed higher concentrations of total LDL, small LDL particles, and LDL compositions with smaller LDL size. The higher level of small LDL concentration was associated with a high Gensini score (B = 0.058, p = 0.024). Compared with the ε3 group, the risk of increased branch lesions in the ε2+ group was lower (OR = 0.416, p = 0.027); (4) Conclusions: The specific allele of APOE being expressed can affect the severity of CHD by altering components of the lipoprotein profile, such as the concentration of small LDL and LDL size.     

Apolipoprotein B100 analysis in microchip with electrochemical detection

Apolipoprotein B100(apoB-100) is a major protein of the cholesterol-rich low-density lipoprotein(LDL) and reflects a better assessment of total atherogenic burden to the vascular system than LDL.In this work,a simple and sensitive method has been developed to determine picoliter apoB-100s using the PMMA microfluidic chip coupled with electrochemical detection system. This method performs very well with a detectable linear range of 1-800 pg/mL and a detection limit of 1 pg/mL.A real serum sample has further been detected by this microchip-based biosensor.The results show that this kind of method is practicable and has the potential application in clinical analysis and diagnosis.     

Probabilistic kinetic model of slow oxidation of low-density lipoprotein. 3. Hydroperoxide-free initiation

A theoretical model is presented that explains slow copper-induced oxidation of low-density lipoprotein in the systems free of seeded hydroperoxydes. The model is based on the probabilistic kinetic theory, modified to take into account different radical generation rates by oxidized and reduced forms of the metal ions. It is shown that the initiation and progression of the LDL oxidation can take place in any LDL dispersion by metal-induced oxidation of alpha-tocopherol, one of the constituents of LDL, and formation of slowly reacting alpha-tocopheroxyl radical. Selected values of several adjustable parameters define actual temporal profiles of the quantities defining the oxidation process.