Miniaturization of asymmetrical flow field-flow fractionation and application to studies on lipoprotein aggregation and fusion

Asymmetrical flow field-flow fractionation (AsFlFFF), a technique that provides direct measurement of particle size and diffusion coefficient, is converted into miniaturized scale. In comparison with conventional AsFlFFF, the separation of proteins in miniaturized AsFlFFF is achieved within shorter time periods, with smaller sample amounts, and with lower mobile phase consumption. Minimization of the overloading and optimization of the separation efficiency are prerequisites to good results. Miniaturized AsFlFFF is applied to the measurement of particle sizes of high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL). The average hydrodynamic diameters at pH 7.4 in 8.5mM phosphate buffer containing 1mM EDTA and 150 mM NaCl are 8.6+/-0.5, 11.2+/-0.2, 22.1+/-0.7, and 48.9+/-7.5 nm for subgroups HDL3, HDL2, LDL, and VLDL, respectively. In addition, the effect of different factors on the aggregation and fusion of LDL particles is studied. LDL particle sizes are unaffected by the addition of up to 300 mM NaCl and by an increase of the carrier solution pH from 3.2 to 7.4, but treatment of LDL with alpha-chymotrypsin, sphingomyelinase, or copper sulfate leads to the formation of aggregated and fused LDL particles.     

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation

Extremophiles - Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses...     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

Separation of human and animal cells by steric field-flow fractionation

In this work, the feasibility of separating and characterizing cell populations by steric field-flow fractionation (steric FFF) is demonstrated by application to fixed human and avian red cells, fresh blood from several species, and viable HeLa cells. The basis for this work is established by means of a discussion of the role of steric FFF in the broad family of field-flow fractionation techniques. The behavior of steric FFF is then characterized by application to standard polystyrene latex beads and to fixed red blood cells. Studies of these standards and of the other cells noted under various conditions of field strength and flow velocity are used to improve the separation conditions and approach optimization. It is shown that the fixed human and avian red cells can be separated in a time of less than 15 min. In addition, it is shown that HeLa cells maintain their viability after passage through the separation channel.     

Remnant-like lipoprotein particles as risk factors for coronary artery disease in elderly patients.

Although remnant-like lipoprotein particles (RLPs) are known to be atherogenic, the relationship between serum RLP-cholesterol (RLP-C) level and coronary artery disease (CAD) has not as yet been evaluated. This clinical study was aimed at investigating the pathological significance of serum RLP-C among several coronary risk factors with a clear focus on elderly patients. We took fasting venous blood samples to determine lipid profiles including RLP-C from 188 patients with angiographically identified CAD and 68 control patients. Overall analysis showed that the RLP-C/HDL-C ratio was higher in both single-vessel CAD group (n = 67; p < 0.01) and multi-vessel CAD group (n = 121; p < 0.001) compared to controls. Further, multiple logistic regression analysis indicated that the diabetes, HDL-C and the RLP-C/HDL-C ratio could discriminate CAD patients from controls. In patients younger than 65 years, diabetes, HDL-C, LDL-C and the LDL-C/HDL-C ratio as well as the RLP-C/HDL-C ratio could discriminate CAD. In patients 65 aged years or older, however, diabetes, triglyceride and RLP-C as well as the RLP-C/HDL-C ratio could discriminate CAD, whereas LDL-C and the LDL-C/HDL-C ratio could not. These results led us to believe that the contribution of a given risk factor to the development of CAD in elderly patients may be different from that in younger patients. In elderly patients, RLP-C rather than LDL-C was strongly associated with the development of CAD. Accordingly, serum RLP-C levels may serve as a convenient and reliable index for assessing CAD.     

Asymmetric flow field-flow fractionation with multiangle light scattering detection for characterization of cellulose nanocrystals

Cellulose nanocrystals (CNCs) were analyzed by asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS) detection. Small fractions were collected from the output of the AF4 apparatus for investigation by transmission electron microscopy (TEM). The influence of CNC injection amount, the number of passes through a high-pressure homogenizer, and different CNC sources on the elution behavior and particle size distribution was investigated. The AF4-MALS results on crystal length were compared with those from TEM. Peak distortion and variation in elution profiles with the increase in sample load were observed. Good resolution was obtained when the injection mass varied from 20 to 40 μg, corresponding to injections of 4-8 μL at a starting concentration of ～5 μg/μL; concentrations during the separation process and at the detector were significantly lower. As the number of homogenization treatments increased, the peak shape became narrower and more symmetrical. This indicates a narrowed crystal length distribution, but regardless of source or homogenization treatment, no CNC preparation was as uniform as tobacco mosaic virus, a well-known rigid rod model structure, whose length was found by AF4-MALS to be in agreement with literature values. CNCs derived from cotton contained longer crystals than those derived from microcrystalline cellulose, as shown by both AF4-MALS and TEM techniques. An advantage of AF4-MALS compared to TEM is the ability to sample large numbers of rodlike particles, which is challenging and time-consuming for TEM image analysis, especially without the presorting afforded by AF4. The good TMV results suggest a high degree of accuracy will pertain to the CNC size distribution measurements.     

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.     

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150 mmol L^?1 sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.     

Modification of glass channel walls for separation of biological particles by gravitational field-flow fractionation

In the gravitational field-flow fractionation of complex samples, various interaction and adsorption phenomena can occur in separation channels that influence fractionation and complicate the explanation of resulting fractograms. To overcome these problems, the glass surface was modified to create charge-free, non-adsorbing hydrophilic media for the mild treatment of hydrophilic biological particles. The modification was carried out in two steps: (1) by a simple lacquering of the glass surface with polystyrene diluted in toluene and (2) subsequent adsorption of a detergent layer on polystyrene. Essential suppression of ionic interactions between soluble low-molecular-mass compounds and the channel wall and decreased adsorption effects were demonstrated in separations of blood samples by gravitational field-flow fractionation.     

Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488 nm was recorded on excitation at 385 nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.     

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.     

Characterization of titanium dioxide nanoparticles imprinted for tyrosine by flow field-flow fractionation and spectrofluorimetric analysis

Films based on TiO₂ nanoparticles (NPs) have been successfully used as sensing elements in chemical sensors. TiO₂ colloidal suspensions can be obtained by spontaneous hydrolysis in acidic solutions of Ti(IV) compounds. The obtained TiO₂ NPs can be employed to build up nanostructured films. With the purpose of preparing TiO₂-based nanostructured, imprinted materials as sensing elements for piezoelectric sensors, we obtained TiO₂ NP dispersions by hydrolyzing potassium titanyl oxalate in the presence of a target analyte (tyrosine). Since morphological properties of the synthesized NPs are known to influence the nanostructured film characteristics, an analytical strategy to characterize such colloidal systems can combine a size-based separation method with spectroscopic analysis to correlate the particle size distribution (PSD) with the particle-target interaction properties able to determine the sensing efficiency.In this work, we present the characterization of colloidal tyrosine-TiO₂ NP systems by flow field-flow fractionation (FlFFF) with online, UV/Vis absorption detection and offline fluorescence analysis. FlFFF eliminates the possible contribution of free tyrosine to the absorption and fluorescence properties of the NPs. FlFFF also fractionates NPs on a size basis. Particle size distribution (PSD) profiles of the fractionated NPs are then obtained by conversion of the multi-wavelength UV/Vis fractograms. Size of the fractionated NPs is finally related to fluorescence properties of the collected NPs fractions. Good correlation between the fluorescence intensity, which is proportional to the tyrosine uptake, and the FlFFF-based, NP mass-size frequency distribution finally confirms the existence of tyrosine-TiO₂ NP interaction.     

Branching features of amylopectins and glycogen determined by asymmetrical flow field flow fractionation coupled with multiangle laser light scattering

The aim of this work was to characterize starch polysaccharides using asymmetrical flow field flow fractionation coupled with multiangle laser light scattering. Amylopectins from eight different botanical sources and rabbit liver glycogen were studied. Amylopectins and glycogen were completely solubilized and analyzed, and high mass recoveries were achieved (81.7-100.0%). Amylopectin Mw, RG, and the hydrodynamic coefficient nuG (the slope of the log-log plot of RGi vs Mi) were within the ranges 1.05-3.18 x 10(8) g mol(-1), 163-229 nm, 0.37-0.49, respectively. The data were also considered in terms of structural parameters. The results were analyzed by comparison with the theory of hyperbranched polymers (Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953; Burchard, W. Macromolecules, 1977, 10, 919-927). This theory, based upon the ABC model, has been shown to underestimate the branching degrees of amylopectins. However, quantitative agreement with the data in the literature was found for amylopectins when using the ABC model modified by the introduction of a multiplying factor, determined from previously described amylopectin structures in terms of the number of branching point calculations.     

Super-ego in patients with coronary artery disease

In this paper, we explored the super-ego ofpatients with coronary artery disease. Research results have confirmed the initial hypothesis that a significant number of patients with coronary artery disease has rigorous super-ego. Among patients with coronary artery disease (N=50), and control group (N=50), we have found significant differences in the quality of super-ego and ego attitude towards the demands of the super-ego. The results of this research contribute to understanding the impact of psychological factors in coronary artery disease.     

Effect of statins on coronary blood flow after percutaneous coronary intervention in patients with stable coronary artery disease

Aims

Statins have favourable effects on the vascular system. However, few data are available regarding the effect of these drugs on patients undergoing percutaneous coronary intervention (PCI). We sought to determine the impact of prior statin use on coronary blood flow after PCI in patients with stable coronary artery disease (CAD) by using the corrected thrombolysis in myocardial infarction (TIMI) frame count (CTFC).

Methods

A total of 80 consecutive eligible patients (mean age: 60 ± 7 years, 65?% male) with the diagnosis of stable CAD who were hospitalised for elective PCI were retrospectively enrolled in our study. The study population was divided into two groups according to statin use at least 6 months before PCI. Group 1 comprised of 51 patients (67?% male; mean age: 58 ± 4 years) taking statins and group 2 comprised of 29 patients (62?% male; mean age: 60 ± 3 years) not taking statins. PCI was applied to de novo type A lesions. CTFC was calculated for the treated vessels at baseline and after PCI.

Results

The two groups had similar characteristics in terms of age, sex, concomitant medications, lesion characteristics, pre-procedural CTFC, lipid parameters, and risk factors for CAD. Post-PCI CTFC (16 ± 3 vs. 22 ± 5, p = 0.01) and hs-CRP (2.1 ± 0.7 mg/l vs. 6.1 ± 2 mg/l, p = 0.01) in patients receiving statins before PCI were significantly lower than in patients without statin therapy. Multiple logistic regression analysis showed that statin pre-treatment (OR 2.5, 95?% CI 1.2 to 3.8, p < 0.001) and hs-CRP level (OR 1.8, 95?% CI 1.2 to 2.4, p = 0.001) were independent predictors of post-PCI CTFC.

Conclusions

In patients with stable CAD undergoing PCI, receipt of long-term statin therapy was associated with improvement in epicardial perfusion after PCI.

     

Two dimensional (pI & ds) separation of phosphorylated proteins by isoelectric focusing/asymmetrical flow field-flow fractionation: application to prostatic cancer cell line

This study demonstrates the use of on-line isoelectric focusing/asymmetrical flow field-flow fractionation (IEF-AF4), a non-gel based high speed two dimensional (isoelectric point and hydrodynamic diameter) protein separation device used for the isolation/separation of phosphoproteins. IEF-AF4 performance was evaluated by first fractionating α-casein molecules at different pIs and sizes. Collected proteins were analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS(n)) to determine various isoforms of the phosphopeptides as well as the relative ratio of phosphorylated and unmodified peptides. A narrow pH cut (ΔpH=0.5) of carrier ampholyte was used in IEF-AF4 to finely resolve phosphoproteins by pI. When the channel lane of multilane AF4 became acidic, the relative ratio of phosphorylated to unmodified or less phosphorylated peptides increased. The current method was applied to prostate cancer cell lysates to demonstrate that IEF-AF4 can examine the relative abundances of specific phosphoproteins, known as biomarkers, in prostate cancer. While affinity-based enrichment methods remove unmodified peptides, IEF-AF4 offers intact phosphoprotein separation at the protein level without removing unmodified proteins. IEF-AF4 enables quantitative analysis without isotope labeling.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.