Biomedical subjects

Csaba Horváth

Publications and source records attributed to Csaba Horváth.

11 recordsLinked to original sources

PubMed2003

Elution-modified displacement chromatography coupled with electrospray ionization-MS: on-line detection of trace peptides at low-femtomole level in peptide digests.

Elution-modified displacement chromatography (EMDC) was employed to achieve peptide separations with high efficiency. On-line ESI-MS and ESI-MS/MS measurements showed enrichment and detection of kemptide, a protein kinase A peptide substrate, at low femtomole levels when it was added as a trace marker component to a tryptic digest of bovine serum proteins or to a human growth hormone peptide digest at concentration ratios between 1:10(5) and 1:10(6). In another EMDC separation, five peptides were detected in a mixture containing 20 fmol of human growth hormone tryptic digest mixed with the bovine serum protein digest. We found that EMDC facilitated rapid detection and sequence analysis of trace peptides at levels of approximately 0.5 fmol/microL in complex peptide mixtures with a wide dynamic concentration range. Accordingly, the detection of kemptide by EMDC was found to be 3-4 orders of magnitude more sensitive than that attained in conventional linear elution chromatography separations performed with the same peptide loads. Kemptide was phosphorylated in vitro and was detected along with its neutral loss product in peptide mixtures at low femtomole levels. EMDC enabled both detection and amino acid sequence determination on trace levels of phosphorylated and other posttranslationally modified peptides, suggesting that the technique may be useful for proteomics applications where detection and analysis of trace level peptides are problematic.

Animals↗

PubMed2003

Capillary electrophoresis of proteins in dextran-coated columns.

A simple coating technique by using uncross-linked dextran has been developed for fused-silica capillaries to be used in capillary electrophoresis of basic proteins. The capillaries were first silanized with a heterobifunctional silane (gamma-aminopropyltriethoxylsilane), which served as a coupling agent between the capillary inner wall and the polysaccharide coating. Dextran of high molecular mass (about 70 kDa) was activated with 1,1'-carbonyldiimidazole. Then the activated dextran was coupled to the primary amino groups that were anchored onto the inner wall of the silanized capillaries. The residual reactive groups on the dextran were further substituted by neutral functions in a coupling reaction with excess ethanolamine. By using dimethyl sulfoxide (DMSO) rather than aqueous buffer as the reaction medium, the extent of substitution was improved by minimizing the residual reactive groups at the surface. Since they are ionogenic, the electrosmotic flow in the system is relatively low. The chemically bound dextran coating showed good reproducibility and stability. In electrophoretic experiments basic proteins were separated with high efficiency by use of the dextran-coated fused-silica capillary columns. The main advantage of the method described here is that both polysaccharide activation and amine-coupling reactions were carried out under mild conditions at room temperature without catalysts. For this reason, the method is recommended to coat the inner wall of microfluidic separation channels which would not tolerate a harsh treatment.

Dextrans↗

PubMed2003

Slight decrease in bone mineralization in cow milk-sensitive children.

BACKGROUND: Patients with cow milk allergy (CMA) are potentially at risk for osteopenia because their milk-free diet usually contains a low calcium content. In our study, different parameters of bone mineralization in children with CMA were investigated. PATIENTS AND METHODS: Twenty-seven CMA patients (mean age, 4.3 years; range, 3-8 years) were enrolled in the study. During a mean milk-free diet period of 11.8 months, children were fed extensively hydrolyzed or soy-based formulas. After a milk challenge test, 7 patients showed allergic symptoms, and the other 20 children had transient CMA. From the sera of all patients, the levels of sodium, potassium, chloride, calcium, phosphate, and magnesium ions, as well as alkaline phosphatase (AP), parathyroid hormone (PTH), osteocalcin, and beta-crosslaps, were determined. These values were compared with those of 20 healthy age-matched controls. Bone mineral density was measured as well. RESULTS: The AP and PTH concentrations were higher in CMA patients than in the control group (AP: 610.2 U/L vs. 499.7 U/L, P < 0.01; PTH: 1.56 pmol/L vs. 0.83 pmol/L, P< 0.03), but all values were in the normal range. The osteocalcin concentration was similar in both groups, and the beta-crosslaps concentration was lower in CMA patients than in controls (0.92 vs. 1.47 ng/mL, P< 0.001). The mean Z score of bone mineral density in patients with CMA was -0.6. In 10 cases, the Z score was less than the -1 SD value. On the basis of the Z score, CMA patients were divided into two groups. The PTH concentration was significantly elevated in the group with lower Z score (2.24 pmol/L vs. 1.16 pmol/L; P< 0.03). CONCLUSION: The results suggest that, in children with CMA who are on a cow milk-free diet, slight disturbances of bone mineralization can be observed by osteodensitometry.

Alkaline Phosphatase↗

PubMed2002

Selective enrichment of low-abundance peptides in complex mixtures by elution-modified displacement chromatography and their identification by electrospray ionization mass spectrometry.

Trace components were selectively enriched and detected in the tryptic digest of recombinant human growth hormone using elution-modified displacement chromatography, a hybrid technique combining features of elution and displacement chromatography. Based on the retention behavior of sample components in the elution mode, rapid and selective trace enrichment and high-resolution separation was achieved in a single step by utilizing appropriate combinations of an eluent such as aqueous acetonitrile with the displacer. Mass spectral and chromatographic analysis of displacement zones revealed up to 400-fold enhancement of the concentration of some low-abundance sample components. Potential application of this technique in proteomics to augment the sensitivity of LC-MS and 2-D gel electrophoretic approaches for the detection of biologically important low-abundance species is discussed.

Amino Acid Sequence↗

PubMed2002

Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres.

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.

Cation Exchange Resins↗

PubMed2002

[The role of adrenal and gonadal hormones in the pathogenesis of autoimmune polyarthritis].

A growing body of recently published results suggest the role of adrenal androgens in the onset and development of chronic inflammatory process due to autoantigens. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEA)--the major androgen products of the adrenal gland--have immunosuppressive effect inhibiting interleukin-6 production and substantially determining acute phase reaction. Decreased serum levels of DHEA and DHEAS has been observed in most of autoimmune diseases. Recent data suggest that adrenal hypoandrogenism comes from disturbed neuroendocrine, regulation due to hypothalamic effect of the inflammatory cytokines. On the other side, decreased adrenal androgen activity negatively influences the anabolic tonus of steroid hormone system while a relative enhancement of catabolic pressure occurs by the glucocorticoids. Moreover, the hypothalamus-hypophysis-gonadal axis can also be involved, resulting shifts in serum levels of prolactin, estrogens and gonadal androgens. All these hormonal changes can be summarised in decreasing the immunosuppressive tonus. This hypothesis connects the endocrine dysregulation with the development of autoimmune disorders. The new results promise not only a basically different theory of chronic inflammation but they will permit using new diagnostic tools as well as inducing substantially new and more effective therapeutic approaches.

Androgens↗

PubMed2002

Capillary electrochromatography of peptides on a column packed with tentacular weak cation-exchanger particles.

Silica-based, tentacular weak cation-exchanger particles were prepared for use as the stationary phase in the separation of positively charged sample components by capillary electrochromatography (CEC). Silica beads were first silanized with 3-(trimethoxysilyl) propyl methacrylate that served as a heterobifunctional linker, which reacted with 2-acrylarmidoglycolic acid in a second step by radical polymerization in aqueous solution. Baseline separation of basic peptides with good column efficiency was obtained on packed capillary columns by isocratic elution CEC with NaCl as the mobile phase modulator. The retention mechanism in the electrochromatographic process was studied by examining the effect of salt concentration on the migration behavior of the peptides. The chromatographic retention factor k'(lc) for charged sample components in the electrochromatographic process was estimated on the assumption that the overall migration rate of a charged migrant can be taken as the sum of the rate of chromatographic elution and the rate of electrophoretic migration. The estimated k(lc) values from experimental results were plotted against the molal salt concentration on a double logarithmic scale. The linear correlation is in good agreement with the prediction by the theory on the basis of traditional ion-exchange chromatography. The comparison of CEC results, obtained with open tubular and packed capillary columns having the same retentive functions as the stationary phase, supports the notion that variation of the phase ratio in the column offers an additional means to modulate the electrochromatographic migration behavior.

Cation Exchange Resins↗

PubMed2002

Preparation of monodisperse porous polymethacrylate microspheres and their application in the capillary electrochromatography of macrolide antibiotics.

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were prepared by a simple one-step dispersion polymerization process. Examination of the polymeric microspheres showed that they had a mean particle diameter of 3 microm and dual pore size distribution with mean pore diameters of 300 and 800 A. The microspheres were functionalized by introducing quaternary ammonium/octadecyl groups to obtain positively charged beads in a wide pH range. The functionalized beads were packed into fused-silica capillary having 50 microm inner diameter and used to separate erythromycin derivatives by capillary electrochromatography (CEC). These samples require gradient elution when separated by high-performance liquid chromatography (HPLC) or micro-HPLC, but with the new columns isocratic elution suffices for their separation by CEC. The column efficiency ranged from 40,000 to 50,000 theoretical plates.

Anti-Bacterial Agents↗

PubMed2002

Streaming potential in open and packed fused-silica capillaries.

The surface properties of novel stationary phases in packed and open tubular columns for capillary electrochromatography (CEC) were examined by measuring the streaming potential in a home made apparatus. The surfaces investigated include materials such as porous styrenic sorbents and octadecyl-silica as well as fused-silica tubing, in both raw and surface modified forms. Functionalization of the surface was carried out, for instance, by reductive amination or organosilane grafting on to capillary inner wall. The dependence of the streaming potential on pH was examined with aqueous solutions in the pH range from 2.5 to 9.0. Electrokinetic properties of 50 microm I.D. fused-silica capillaries have been determined by both streaming potential and electrosmotic flow measurements. Both methods gave similar pH profiles of the zeta-potential and the isoelectric points. This confirms the viability of our approach to evaluate the specific charged groups of the packing which is one of the important factors influencing electrosmotic flow (EOF) velocity and protein adsorption during a chromatographic run. In addition to bare silica capillaries, styrenic monolithic columns with different surface functionalities, which have been extensively used in our laboratory for CEC separation of peptides and proteins, were employed for comparison of two methods. Plots of zeta potential as a function of percent ACN show a complex behavior, indicating that zeta potential cannot be predicted simply from binary mixture solvent properties. It is demonstrated that the evaluation of the zeta potential by the streaming potential method is nondestructive, relatively fast, without untoward effects introduced by Joule heating and yet another means for the characterization of the surfaces under conditions employed in CEC.

Adsorption↗

PubMed2002

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components.

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.

Algorithms↗

PubMed2002

Chromatographic and electrophoretic migration parameters in capillary electrochromatography.

Chromatographic retention factor, k', as defined in high-performance liquid chromatography (HPLC) in terms of the migration times of the separand and the inert tracer, has limited applicability to capillary electrochromatography (CEC) when both chromatographic and electrophoretic processes determine the magnitude of the overall migration rates of the separands. This situation is unlike that in HPLC, where k' serves as a useful peak locator for the various sample components, as well as, provides thermodynamic insights into the interactions between the components and the stationary phase. Most publications have borrowed the definition of k' from HPLC and applied it on CEC. However, due to the dual separation mechanisms that are in action in CEC, the system is significantly complicated in comparison to that of HPLC. This paper discusses the impossibility of defining with a k' which would have all the attributes that it has in regular chromatography. The interplay of the two separation mechanisms in determining the overall migration process in CEC is discussed and the various definitions of the electrochromatographic retention factor are presented.

Chromatography, High Pressure Liquid↗