Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation

Granulocytes are large, stiff viscoelastic cells that adhere naturally to the vascular endothelium. On their passage through the capillary network they have to be deformed, and recent evidence indicates that they may impose a significant hemodynamic resistance. The entry time of granulocytes into capillaries is about three orders of magnitude longer than that for red cells. Inside the capillary the granulocytes move with a lower velocity than red cells. Under conditions when the capillary perfusion pressure is reduced and/or elevated levels of inflammatory products are present that increase the adhesion stress to the endothelium, granulocytes may become stuck in the capillary. In such a situation, the granulocytes form a large contact area with the capillary endothelium, they obstruct the lumen, and they may initiate tissue injury. After the restoration of the perfusion pressure the granulocytes may not be removed from the capillary owing to the adhesion to the endothelium. Capillary plugging by granulocytes appears to be the mechanism responsible for the no-reflow phenomenon, and together with oxygen free radical formation and lysosomal enzyme activity may constitute the origin for ischemic injury as well as other microvascular occlusive diseases.     

Microscale separation and analysis

There is a recent and growing interest in microscale separation and analysis, a result of advantages of miniaturization such as rapid separation times, high performance and throughput, reduced costs, and the possibility of system integration and multiplexing. Adopting the concepts of conventional capillary electrophoresis, capillary electrochromatography, micellar electrokinetic chromatography and various sample preparation techniques to microchip format, in conjunction with the integration of different analysis steps into a monolithic system, have opened new levels in performance, functionality and throughput. This review summarizes the recent advances in the field of microfabricated separation devices for genomics, proteomics and high-throughput screening applications, also addressing system integration and micropreparative functionalities.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides

Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper.     

GPIHBP1, an endothelial cell transporter for lipoprotein lipase

Interest in lipolysis and the metabolism of triglyceride-rich lipoproteins was recently reignited by the discovery of severe hypertriglyceridemia (chylomicronemia) in glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1)-deficient mice. GPIHBP1 is expressed exclusively in capillary endothelial cells and binds lipoprotein lipase (LPL) avidly. These findings prompted speculation that GPIHBP1 serves as a binding site for LPL in the capillary lumen, creating "a platform for lipolysis." More recent studies have identified a second and more intriguing role for GPIHBP1-picking up LPL in the subendothelial spaces and transporting it across endothelial cells to the capillary lumen. Here, we review the studies that revealed that GPIHBP1 is the LPL transporter and discuss which amino acid sequences are required for GPIHBP1-LPL interactions. We also discuss the human genetics of LPL transport, focusing on cases of chylomicronemia caused by GPIHBP1 mutations that abolish GPIHBP1's ability to bind LPL, and LPL mutations that prevent LPL binding to GPIHBP1.     

Analysis of disease-causing genes and DNA-based drugs by capillary electrophoresis towards DNA diagnosis and gene therapy for human diseases

Rapid progress in the Human Genome Project has stimulated investigations for gene therapy and DNA diagnosis of human diseases through mutation or polymorphism analysis of disease-causing genes and has resulted in a new class of drugs, i.e., DNA-based drugs, including human gene, disease-causing gene, antisene DNA, DNA vaccine, triplex-forming oligonucleotide, protein-binding oligonucleotides, and ribozyme. The recent development of capillary electrophoresis technologies has facilitated the application of capillary electrophoresis to the analysis of DNA-based drugs and the detection of mutations and polymorphism on human genes towards DNA diagnosis and gene therapy for human diseases. In this article the present state of studies on the analysis of DNA-based drugs and disease-causing genes by capillary electrophoresis is reviewed. The paper gives an overview of recent progress in the Human Genome Project and the fundamental aspects of polymerase chain reaction-based technologies for the detection of mutations and polymorphism on human genes and capillary electrophoresis techniques. Attention is mainly paid to the application of capillary electrophoresis to polymerase chain reaction analysis, restriction fragment length polymorphism, single strand conformational polymorphism, variable number of tandem repeat, microsatellite analysis, hybridization technique, and monitoring of DNA-based drugs. Possible future trends are also discussed.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Tissue proteomics using capillary isoelectric focusing-based multidimensional separations

The capabilities of capillary isoelectric focusing-based multidimensional separations for performing proteome analysis from minute samples create new opportunities in the pursuit of biomarker discovery using enriched and selected cell populations procured from tissue specimens. In this article, recent advances in online integration of capillary isoelectric focusing with nano-reversed phase liquid chromatography for achieving high-resolution peptide and protein separations prior to mass spectrometry analysis are reviewed, along with its potential application to tissue proteomics. These proteome technological advances combined with recently developed tissue microdissection techniques, provide powerful tools for those seeking to gain a greater understanding at the global level of the cellular machinery associated with human diseases such as cancer.     

Affinity capillary electrophoresis: important application areas and some recent developments

Affinity capillary electrophoresis (ACE) is a broad term referring to the separation by capillary electrophoresis of substances that participate in specific or non-specific affinity interactions during electrophoresis. The interacting molecules can be found free in solution or can be immobilized to a solid support. Every ACE mode has advantages and disadvantages. Each can be used for a wide variety of applications. This paper focuses on applications that include purification and concentration of analytes present in diluted solutions or complex matrices, quantitation of analytes based on calibration curves, and estimation of binding constants from direct and derived binding curves based on quantitation of analytes or on analyte migration shifts. A more recent chemicoaffinity strategy in capillary electrophoresis/capillary electrochromatography (CE/CEC) termed molecular imprinting (`plastic antibodies') is discussed as well. Although most ACE studies are aimed at characterizing small-molecular mass analytes such as drugs, hormones, and peptides, some efforts have been pursued to characterize larger biopolymers including proteins, such as immunoglobulins. Examples of affinity interactions that have been studied are antigen–antibody, hapten–antibody, lectin–sugar, drug–protein, and enzyme–substrate complexes using ultraviolet, laser-induced fluorescence, and mass spectrometer detectors. This paper also addresses the critical issue of background electrolyte selection and quantitation of analytes. Specific examples of bioaffinity applications are presented, and the future of ACE in the biomedical field is discussed.     

Capillary electrophoretic assay and purification of cylindrospermopsin, a cyanobacterial toxin from Aphanizomenon ovalisporum, by plant test (blue-green Sinapis test)

Toxic cyanobacteria are known to produce cyanotoxins, toxic secondary metabolites. In recent years the cylindrospermopsin (tricyclic guanidinyl hydroxymethyluracil)-producing organisms Aphanizomenon ovalisporum, Cylindrospermopsis raciborskii, and Umezakia natans have been inhabiting polluted fresh waters. Cylindrospermopsin, a potent hepatotoxic cyanotoxin, has been implicated in cases of human poisoning as well. This study describes the isolation and purification of cylindrospermopsin from A. ovalisporum with the help of a slightly modified Blue-Green Sinapis Test, a plant test suitable for determining the cyanotoxin content of chromatographic fractions besides plankton samples. The recent modification, using microtiter plates for the assay, improves the method and reduces the amount of sample needed for the assay. This approach proved that plant growth and metabolism, at least in the case of etiolated Sinapis alba seedlings, are inhibited by cylindrospermopsin. The establishment of capillary electrophoresis of cylindrospermopsin and consideration of the results reported here lead us to the expectation that capillary electrophoresis of cylindrospermopsin may be a powerful and useful analytical method for investigating cyanobacterial blooms for potential cylindrospermopsin content and toxicity. Confirmation of chemical identity of the purified compound is performed by UV spectrophotometry, NMR, and MALDI-TOF.     

Analysis of glycoprotein heterogeneity by capillary electrophoresis and mass spectrometry

Glycosylation is a complex posttranslational modification that can result in extensive heterogeneity for recombinant glycoproteins produced by eukaryotic systems. The carbohydrate moiety of a recombinant glycoprotein may affect the immunogenicity, half-life, bioactivity, and stability of a potential therapeutic product. Regulatory authorities such as the US Food and Drug Administration demand increasingly sophisticated carbohydrate analysis to ensure product characterization, batch-to-batch consistency, and stability. The advent of new technologies for analysis of biopolymers by capillary electrophoresis and mass spectrometry has revolutionized strategies for recombinant protein characterization. In particular, recent advances in matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry now permit relatively rapid and detaned assessment of glycoprotein and oligosaccharide structure. In this article, we describe some applications of capillary electrophoresis and mass spectrometry to monitor the glycosylation associated with a model recombinant glycoprotein, human interferon-γ.     

Pulmonary capillary and permeability during hemorrhagic shock

In previous experiments from this laboratory horseradish peroxidase was used to study the structural and functional characteristics of the normal canine pulmonary capillary membrane. The present study used the same technique to try to determine if any change occurred in the pulmonary capillary as a result of hemorrhagic shock. We found that hemorrhagic shock caused a fall (5 expt) or no change (2 expt) in estimated pulmonary transcapillary pressure based on Starling's equation. However, lymphatic flow from the lungs increased. Estimated of filtration coefficients showed a highly significant increase (P less than 0.01) during the hypotensive period. Pulmonary lymphatic protein concentrations were not altered, indicating that water and protein continued to traverse the membrane in the same proportions as under normotensive conditions. These data are consistent with recent observations of minimal changes in the intercellular junctions of the capillary endothelium following hemorrhagic shock made independently on lung tissue from these experiments.     

A mathematical theory of capillary exchange as a function of tissue structure

A mathematical theory of the process of the exchange of substances between the blood in the capillaries of a homogeneous tissue and the extracellular space, and between the extracellular space and the cells is developed. An ideal geometry of the tissue is assumed, based to some extent on recent anatomical work concerning the functional distinction between two types of capillaries, the arteriolo-venular and the true capillaries. Equations are developed relating the concentration in the arterial blood to the mean capillary concentration, the concentration at the wall of the capillary in the extracellular space, and the average concentration in the extracellular space, and also relating the cellular concentration to the average extracellular concentration. The solutions of the equations are given for certain special cases and numerical results obtained. It is shown that the average extracellular concentration is a sensitive function of the permeability of the capillary wall and also is strongly influenced by the diffusion coefficient of the extracellular space. Furthermore, it is shown that the speed with which the average extracellular concentration approaches the steady state is largely a function of the permeability of the capillary wall.     

Blood flow in the capillary bed

From arteries to veins, the blood has to go through the ‘capillary’ blood vessels. These blood vessels are so small that often their diameter is smaller than that of the red blood cells. Intimate interactions occur, therefore, between the blood cells and the blood vessels.

A general survey of recent works on capillary blood flow is given in this article. Some details are presented for two problems: the problem of deformation of the flexible red blood cells, their motion in the capillary blood vessels, and the pressure drop due to the red cell blood vessel interaction; and the problem of the flow of plasma ‘bolus’ between neighboring red cells.

The solution supplies many details about the microcirculation phenomenon. Taken together, a method is offered for the calculation of pressure drop in the capillary as a function of various physical parameters: the red cell volume per unit blood volume, (hematocrit), the ratio of the cell diameter to the blood vessel diameter, the ratio of the length of the blood vessel to its length, the volume of individual red cells, and a parameter relating the cell membrane elasticity, plasma viscosity and the cell velocity.     

Biophysical basis of glomerular permselectivity

Summary The mammalian glomerular capillary wall normally restricts the transmural passage of plasma proteins while offering little resistance to the filtration of water and small solutes. The basis for this selectivity has been explored extensively in recent years, through clearance measurements of endogenous (mainly albumin, transferrin, and immunoglobulins) and exogenous (horseradish peroxidase) proteins, and a variety of nonprotein polymers such as dextrans and polyvinylpyrrolidone. In conjunction with efforts to localize particulate and soluble tracers by high resolution ultrastructural techniques, such measurements have now made it possible to define the determinants of the glomerular filtration of macromolecules in terms of discrete structural barriers as well as such biophysical influences as hemodynamics and the molecular size- and charge-selective characteristics of the capillary wall.These experimental approaches have been aided greatly by the development of theoretical models that enable investigators to describe macromolecular filtration in terms of hydrodynamic principles applied to isoporous membranes. Although the initial models failed to consider the important role of membrane fixed negative-charge characteristics in influencing protein filtration, this shortcoming has led to the recent introduction of a theoretical model that also takes this factor into consideration. The aim of this brief review is to summarize these various theoretical approaches to the understanding of glomerular permselectivity and, wherever possible, to cite specific tests of these theories based on experimental studies in humans and animals.     

3D-biohybrid systems: applications in drug screening

Biotechnology demands powerful methods for the functional characterisation and monitoring of molecular alterations in tissues in response to various stimuli. Currently, cellular biosensors provide information about cell and tissue internal transduction pathways. In this article, recent biosensor systems are briefly described and the use of 3D tissue aggregates as recognition elements is discussed. An example of an innovative approach for drug testing using 3D heart muscle aggregates, as well as tumor models, positioned in capillary systems for electrical potential recording and impedance measurement is described. The effectiveness of drugs and therapies can be tested and monitored in a short time using such biohybrid sensors.     

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance.     

Bioanalysis of drugs by liquid-phase microextraction coupled to separation techniques

The demand for automation of liquid-liquid extraction (LLE) in drug analysis combined with the demand for reduced sample preparation time has led to the recent development of liquid-phase microextraction (LPME) based on disposable hollow fibres. In LPME, target drugs are extracted from aqueous biological samples, through a thin layer of organic solvent immobilised within the pores of the wall of a porous hollow fibre, and into an microl volume of acceptor solution inside the lumen of the hollow fibre. After extraction, the acceptor solution is subjected directly to a final analysis either by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), mass spectrometry (MS), or capillary gas chromatography (GC) without any further treatments. Hollow fibre-based LPME may provide high enrichment of drugs and excellent sample clean-up, and probably has a broad application potential within the area of drug analysis. This review focuses on the principle of LPME, and recent applications of three-phase, two-phase, and carrier mediated LPME of drugs from plasma, whole blood, urine, and breast milk.