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Biomedical subjects

C Horváth

Publications and source records attributed to C Horváth.

At least 19 recordsLinked to original sources
PubMed2002

Lack of association between calcium-sensing receptor gene "A986S" polymorphism and bone mineral density in Hungarian postmenopausal women.

Calcium-sensing receptor (CaSR) is an attractive candidate gene for osteoporosis susceptibility. The CaSR "A986S" genotype has been shown to have an effect on serum calcium. Recently, an association has been reported between the CaSR gene A986S polymorphism and bone mineral density in healthy white girls. In this study, we examined whether CaSR gene A986S polymorphism is associated with decreased bone mass in 230 Hungarian postmenopausal women. From this cohort, 108 osteoporotic patients were compared with 122 healthy control women. Bone mineral density (BMD) was measured at the lumbar spine (L2-4) and femoral neck using dual-energy X-ray absorptiometry. Allele-specific polymerase chain reaction was used to amplify A986S polymorphisms of the CaSR gene. We found no difference in the distribution of different alleles or genotypes between groups (p = 0.762). No significant effect of CaSR genotype on BMD was observed either in the whole population or in the subgroups. Our data do not support the idea that CaSR gene A986S polymorphism has an impact on bone mass.

Aged↗
PubMed2001

[New intraoperative diagnostic methods in parathyroid surgery: intraoperative measurement of parathyroid hormone].

In thirty-seven patients undergoing parathyroidectomy from November 1999 to July 2000 with diagnosis of primary hyperparathyroidism the intraoperative intact parathyroid hormone level were studied. Preoperative samples of blood were taken from a peripheral vein before the beginning the operation. The second sample was taken 5 minutes after removal of parathyroid adenoma. The plasma intact parathyroid hormone concentration was measured by quick IRMA method. The preoperative high PTH levels reduced after excision of adenoma to the mean 18%. 33 patients had normal PTH levels after 5 minutes of removing. The total measuring time of quick PTH assay was about 30 minutes. The QPTH assay was a quantitative adjunct for the surgeon to ensure a successful parathyroidectomy. When the intraoperative QPTH level is not reduced to 50%, then the exploration should be continued and the probability of reoperation is decreased. They propose the application of the routine intraoperative measurement of QPTH level in all parathyroidectomy.

Adenoma↗
PubMed2001

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature.

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2001

[Follow-up study of bone mineral density in postmenopausal patients with primary biliary cirrhosis].

Osteopenia is a common complication in primary biliary cirrhosis (PBC). In this follow-up study the authors investigated the metabolic bone disease in postmenopausal PBC patients. 17 Ca and vitamin D supplemented, postmenopausal female patients with PBC (stage II-IV, age: 41-84, mean: 52, each AMA M2 positive, without ascites) were followed-up for an average of 6.3 years. Bone mineral density (BMD) was measured yearly by dual energy x-ray absorptiometry (XR26, Norland) in lumbar spine (L2-4), femoral neck (FN), and radius BMC by single photon absorptiometry. Urinary pyridinoline/creatinine (Pyr/c) and deoxypyridinoline/creatinine ratio (D-Pyr/c) by HPLC, 25-OH-D3 level and standard liver function tests were monitored in all patients. At the beginning the BMD was decreased in 7 out of 17 patients (T-score < -2.5). The mean BMD was 0.885 SD +/- 0.26 g/cm2 in L2-4, 0.725 +/- 0.16 g/cm2 in FN and the BMC 0.703 +/- 0.14 g/cm in the radius. During follow-up the rate of annual bone loss was increased in patients with osteoporosis at the start of this study. There was a correlation between the urinary Pyr/c and D-pyr/c values and the annual rate of bone loss in patients with PBC (r: -0.79; p < 0.01). In patients with severe osteoporosis at the time of the diagnosis of PBC a more pronounced progression of bone loss was observed during the follow-up period.

Absorptiometry, Photon↗
PubMed2001

Enhancement of electroosmotic flow in capillary electrochromatography.

A major impediment to enhancing the speed of separation in capillary electrochromatography (CEC) is the upper limit on the electroosmotic flow (EOF) velocity by the maximal zeta potential of the chromatographic surface. Here, a new approach to speeding up EOF, suggested by Yang and El Rassi (Electrophoresis 1999, 20,18-23), is examined critically. It entails the use of a tandem arrangement of a separating column and an auxiliary column, the sole function of which is to boost EOF velocity in the separating column and thus facilitate faster analysis by CEC. Based on the principle of conservation of mass and current and using experimental data obtained in a wide range of conditions, the flow velocities in the separating and auxiliary columns were evaluated. The results show that an equidiameter open tubular auxiliary column offers a greater enhancement of EOF velocity than a packed column. Nevertheless, within the scope of the experiments the enhancement of EOF velocity by as much as 50% by using open tubular auxiliary columns has been obtained.

Chromatography↗
PubMed2000

Capillary electrochromatography of proteins and peptides with a cationic acrylic monolith.

For the separation of proteins and peptides by capillary electrochromatography (CEC), columns with a monolithic stationary phase were prepared from silanized fused-silica capillaries of 50 microm I.D. by in situ copolymerization of glycidyl methacrylate, methyl methacrylate and ethylene glycol dimethacrylate in the presence of propanol and formamide as porogens. The epoxide groups at the surface of the porous monolith were reacted with N-ethylbutylamine to form fixed tertiary amino functions with ethyl- and butyl-chains. A mixture of ribonuclease A, insulin, alpha-lactalbumin and myoglobin was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and sodium phosphate buffer, pH 2.5. The separation of four angiotensin type peptides by CEC was also achieved under similar conditions. The elution order of proteins was similar to that obtained in reversed-phase chromatography. Plots of the migration factors for proteins and peptides against the acetonitrile concentration exhibit opposite trends. This is most likely due to the greater chromatographic retention and lower electrophoretic migration velocity of proteins than that of peptides in the counterdirectional CEC system. From this it is concluded that the separation is governed by a dual mechanism that involves the complex interplay between selective chromatographic retention and differential electrophoretic migration.

Acetonitriles↗
PubMed2000

Capillary electrochromatography of proteins on an anion-exchanger column.

Capillary electrochromatography (CEC) of proteins was carried out using 50-microm-i.d. fused-silica capillaries packed with 5-microm silica beads having strong anion-exchanger functions attached to hydrophilic spacers at the chromatographic surface. The siliceous microspheres and the capillary innerwall were treated first with a heterobifunctional silanizing agent and reacted subsequently with a vinyl monomer containing quaternary ammonium groups to form a "tentacular" anion exchanger. A mixture of bovine carbonic anhydrase, alpha-lactalbumin, soybean trypsin inhibitor, and ovalbumin was separated using CEC by isocratic elution in the codirectional mode with aqueous phosphate buffer, pH 7.0, containing sodium chloride. The retention mechanism of isocratic CEC for proteins on the anion-exchanger column was illustrated by the results of a study on the effect of salt concentration on the separation. The potential of CEC for protein separation with high resolution was also demonstrated by electrochromatograms of conalbumin and hemoglobin variants. The results shed light on the mechanism of protein separation by isocratic CEC, which is believed to be a combination of chromatographic retention by electrostatic interactions and electrophoretic migration. Assuming that the contributions of the two mechanisms to the overall migration velocity are additive, an electrochromatographic resolution equation was derived and compared to the resolution equation in HPLC to reveal the constituents responsible for the enhancement of resolution by CEC with respect to that in HPLC. The advantage of CEC was also examined by comparing peak capacities in CEC on an, isocratic platform with peak capacities obtained with isocratic and gradient elution HPLC.

Anions↗
PubMed2000

Protein retention in ion-exchange chromatography: effect of net charge and charge distribution.

The charge regulated slab model is used to evaluate the salt dependence of the retention of Staphylococcal nuclease A and its mutants in cation-exchange chromatography. An important feature of this work is that the net charge of the proteins is varied in two different ways: (a) by changing the eluent pH so that the charges are created by protonation and (b) by point mutation at position 116. Since the structure of Staphylococcal nuclease and the mutants are known, the pH dependence of retention data of the different mutants gives detailed insights into the retention mechanism. Experimental results show that the salt dependence of retention is affected more strongly by changes of the eluent pH than by point mutations. This implies that the amino acid in position 116 has only a moderately strong interaction with the stationary phase surface and that a patch on one side of the protein surface is mainly responsible for the electrostatic interaction with the surface.

Chromatography, Ion Exchange↗
PubMed1999

Capillary electrochromatography of proteins and peptides with porous-layer open-tubular columns.

Capillary electrochromatography (CEC) of basic proteins and peptides was carried out with porous-layer open-tubular (PLOT) columns which had a functionalized rugulose polymeric porous layer grafted to the innerwall of 20 microm I.D. fused-silica capillaries. The porous layer was highly crosslinked and prepared by in situ polymerization of vinylbenzyl chloride and divinylbenzene in the presence of 2-octanol as a porogen inside a pretreated fused-silica capillary. The chloromethyl functions at the surface of the porous polymeric support layer were reacted with N,N-dimethyldodecylamine to obtain a positively charged chromatographic surface with fixed C12 alkyl chains. A mixture of lysozyme, cytochrome c, ribonuclease A and alpha-chymotrypsinogen A was separated isocratically by counterdirectional CEC with hydro-organic mobile phases containing acetonitrile and phosphate buffer, pH 2.5. The overall migration behavior of the four proteins was the result of an interplay of chromatographic retention and electrophoretic migration, and was different from that observed in capillary zone electrophoresis or in reversed-phase chromatography under similar conditions. The separation of three basic peptides by CEC also exhibited the same behavior. The stability of the PLOT column was tested by measuring electroosmotic mobility during continual use.

Chromatography, Liquid↗
PubMed1999

Dynamics of capillary electrochromatography. II. Comparison of column efficiency parameters in microscale high-performance liquid chromatography and capillary electrochromatography.

In capillary electrochromatography (CEC) the flow of the mobile phase is generated by electrosmotic means in high electric field. This work compares band spreading measured experimentally in several packed capillaries with electrosmotic flow (EOF) and viscous flow under otherwise identical conditions. The data were fitted to the simplified van Deemter equation for the theoretical plate height, H = A + B/u + Cu, in order to evaluate parameters A and C in each mode of flow in the different columns. The ratio of these two parameters obtained with the same column in microscale HPLC (mu-HPLC) and CEC was used to quantify the attenuation of their contribution to band spreading upon changing from viscous flow (in mu-HPLC) to electrosmotic flow (in CEC). The capillary columns used in this study were packed with stationary phases of different pore sizes as well as retentive properties and measurements were carried out under different mobile phase conditions to examine the effects of the retention factor and buffer concentration. In the CEC mode, the value of both column parameters A and C was invariably by a factor of two to four lower than in the mu-HPLC mode. This effect may be attributed to the peculiarities of the EOF flow profile in the interstitial space and to the generation of intraparticle EOF inside the porous particles of the column packing. Thus, band spreading due to flow maldistribution and mass transfer resistances is significantly lower when the mobile phase flow is driven by voltage as in CEC, rather than by pressure as in mu-HPLC.

Buffers↗
PubMed1999

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography.

Capillary columns with monolithic stationary phase were prepared from silanized fused-silica capillaries of 75 microns I.D. by in situ copolymerization of divinylbenzene either with styrene or vinylbenzyl chloride in the presence of a suitable porogen. The porous monolithic support in this study was used either directly or upon functionalization of the surface to obtain a stationary phase that was appropriate for the separation of peptides by capillary electrochromatography (CEC). The main advantages of monolithic columns are as follows. They do not need retaining frits, they do not have charged particles that can get dislodged in high electric field, and they have relatively high permeability and stability. Whereas such columns are designed especially for CEC, they find application in micro high-performance liquid chromatography (mu-HPLC) as well. Five different porogens were employed to prepare the monolithic columns that were examined for permeability and porosity. The flexibility of fused-silica capillaries was not adversely affected by the monolithic packing and the longevity of the columns was satisfactory. This may also be due to the polymerization technique, which resulted in a fluid-impervious outer layer of the monolith that precluded contact between the fused-silica surface and the liquid mobile phase. For the most promising columns, the conductivity ratios and the parameters of the simplified van Deemter equation, both in mu-HPLC and CEC, were evaluated. It was found that the efficiency of the monolithic columns in CEC was significantly higher than in mu-HPLC in the same way as observed with capillary columns having conventional particulate packing. This is attributed to the relaxation of band-broadening with electroosmotic flow (EOF) with respect to that with viscous flow. It follows then that the requirement of high packing uniformity to obtain high efficiency may also be relaxed in CEC. Angiotensin-type peptides were separated by CEC with columns packed with a monolithic stationary phase having fixed n-octyl chains and quaternary ammonium groups at the surface. Plate heights of about 8 microns were routinely obtained. The mechanism of the separation is based on the interplay between EOF, chromatographic retention and electrophoretic migration of the positively charged peptides. The results of the complex migration process, with highly nonlinear dependence of the migration times on the organic modifier and the salt concentration, cannot be interpreted within the framework of classical chromatography or electrophoresis.

Amino Acid Sequence↗
PubMed1999

Electrosmotic mobility and conductivity in columns for capillary electrochromatography.

Columns employed so far in capillary electrochromatography (CEC) contain both a packed and an open segment with concomitant changes of the electric field strength and the flow velocity at the interface of the two segments in such duplex columns. To take this into account in measuring, processing, and interpreting CEC data, a framework is presented for the evaluation of the conductivity ratio and the interstitial electrosmotic flow (EOF) mobility and their usage as tools for characterizing CEC columns. This is illustrated by experimental data obtained from measurement of the current and the EOF in capillary columns packed with different stationary phases. The current data yielded the ratio of the conductivities of the packed and open segments that has been shown to be useful for the evaluation of the porosity and tortuosity. It is assumed that these important packing characteristics are the same for the flow of current and for the flow of the bulk mobile phase in the CEC column. The EOF mobility in such duplex columns is defined in two different ways. The apparent mobility, which is widely reported at present, is obtained from the length of packed segment, the migration time, and the overall electric field strength. On the other hand, the actual mobility is obtained after taking into account the porosity and tortuosity of the packing as well. Thus, the actual mobility is made independent of the porosity and tortuosity and therefore can be useful to estimate the zeta potential for characterizing the packing surface. Measurements of both the apparent and actual electrosmotic mobilities for a number of different columns have shown that the apparent and actual mobilities are significantly different in their magnitude. For this reason, it is recommended that, instead of the apparent EOF mobility, the actual mobility is used for the characterization of the packing in CEC columns.

Chromatography, Liquid↗
PubMed1999

Effect of perinatal synthetic steroid hormone (allylestrenol, diethylstilbestrol) treatment (hormonal imprinting) on the bone mineralization of the adult male and female rat.

Neonatal treatment with allylestrenol or diethylstilbestrol (DES) reduced the bone mineral content (BMC/bw) of the adult (four months old) female rats, without influencing bone mineral density (BMD/bw). In males these neonatal treatments elevated BMC and BMD alike. Ovariectomy alone decreased BMC and BMD alike; however the neonatal hormone treatments did not influence this reduced value. Ovariectomy of two months old animals increased body weight without the influence of neonatal hormone treatments. In adult males, the body weight was reduced significantly by neonatal DES and non-significantly by neonatal allylestrenol treatment. The experiments call attention to the possible human bone-effects of allylestrenol, which was used in the last decades as medication protecting endangered pregnancies.

Absorptiometry, Photon↗
PubMed1998

Retention in reversed-phase chromatography: partition or adsorption?

A unified framework within the hermeneutics of the solvophobic theory is employed for the treatment of experimental data with nonpolar and weakly polar substances in reversed-phase chromatography (RPC), oil-water partitioning and adsorption on activated charcoal from dilute aqueous solution. This approach sheds light on the energetic similarities between such processes driven by the hydrophobic effect. Among several stationary phase models that have been proposed in the literature for the physical representation of alkyl-silica bonded phases, the isolated solvated hydrocarbon chains model is adopted for the retention in RPC since it represents most closely the stationary phase configuration and is not based a priori on a partition or adsorption mechanism as some other models are for the retention in RPC. Using the fundamental framework of the solvophobic theory, the free energy change per unit nonpolar surface area for octanol-water and hexadecane-water partitioning, retention in RPC as well as adsorption on activated charcoal from dilute aqueous solution at 25 degrees C are evaluated and they are found to be in good agreement with the corresponding experimental data. Furthermore, such quantities are very similar for all the above mentioned processes involving aqueous solution, in contradistinction to the predictions by the lattice theory. From the results it follows that these apparently disparate processes are subject to the same physicochemical principle. The present study demonstrates the capability of the solvophobic theory in describing the energetics of processes involving hydrophobic interactions, and exposes the difficulties in distinguishing between partition and adsorption mechanisms in RPC by using partition models based on the lattice approach. It is concluded that a clear distinction between partition and adsorption in RPC of nonpolar elites is not apparent from thermodynamic analysis.

Adsorption↗
PubMed1998

Capillary electrochromatography of biomolecules with on-line electrospray ionization and time-of-flight mass spectrometry.

Capillary electrochromatography (CEC) is considered a hybrid of liquid chromatography and capillary electrophoresis. It is expected to combine the high peak efficiency of capillary zone electrophoresis with the versatility and loading capacity of HPLC to bring about another high-performance MS-compatible chromatographic system. This paper explores the potential of CEC coupled with the electrospray ionization and time-of-flight mass spectrometry in biochemical analysis. The packed columns used in this study were tapered at the outlet to retain the packing material, thereby obviating the need for an outlet frit. Electrosmotically driven solvent gradients were employed for the separation of phenylthiohydantoin (PTH)-amino acids by reversed-phase chromatography, and a time-of-flight (TOF) mass spectrometer was employed as the detector for the CEC column effluent. The effect of CEC operating parameters, such as gradient shape, column length, and electric field, on the analytical results from the separation and MS detection of a standard mixture of PTH-amino acids was investigated. Particular attention was paid to the effect of sheath flow-rate, sheath composition and mass spectra acquisition rate on the performance of the electrospray TOF-MS.

Amino Acids↗
PubMed1998

Capillary zone electrophoresis at subzero temperatures. III. Operating conditions and separation efficiency.

The advantages of carrying out capillary zone electrophoresis at subambient rather than ambient temperatures are examined. They include the possibility of using higher electric field strength and/or buffer concentration as well as wider capillary lumen without untoward effects of Joule heating on the separation. Furthermore, the separation efficiency is enhanced as molecular diffusivity decreases with temperature. A Beckman P/ACE unit with an auxiliary cooling system was used at temperatures down to -20 degrees C and, based on the dependence of axial temperature gradient on the capillary length and the temperature of the coolant, an average capillary temperature was defined. The day-to-day migration times of proteins were reproducible with an R.S.D. better than 2.3%. The improvements in the separation efficiency upon lowering the temperature from 40 to 1 degree C are illustrated by the electropherograms of four closely related peptides. Generally, the plate efficiency is enhanced at low temperatures and the analysis time is prolonged due to the increased viscosity. However, the current at a fixed electric field also decreases with temperature and the capillary can be shortened to speed up the analysis. With benzenesulfonic acids, the rate of the generation of theoretical plates was two- to three-times higher using a 27 cm long capillary at -15 degrees C than a 47 cm long capillary at 40 degrees C. By using a 180 microns instead of a 50 microns I.D. capillary for the separation of the four peptides, the sample loading could be increased about 15 times without loss of resolution. In the capillary zone electrophoresis of proteins, it was found that protein interactions with the capillary wall can be significantly reduced by increasing buffer concentration at low temperatures where salt mediated hydrophobic interactions are attenuated. This is illustrated by the separation of beta-lactoglobulins A and B in a raw fused-silica capillary using 400 mM sodium borate buffer, pH 8.4. The results suggest that at subambient or even subzero temperatures, the scope of CZE can be extended by several ways so that the benefits are great enough to justify the use of a cooling system and temperature control appropriate for psychroelectrophoresis with fused-silica capillaries.

Buffers↗
PubMed1998

Effect of a predetection open segment in the column on speed and selectivity in capillary electrochromatography.

Columns in capillary electrochromatography (CEC) most commonly have the detection window located immediately after the retaining frit of the packed segment. Here, the properties of "duplex" columns having a predetection open segment between the frit and the detector window are examined with particular regard to the effect of the relative lengths of the packed and open segments on the separation of mixtures containing neutral and charged components. This configuration allows the use of columns with short packed segments in contemporary instruments for rapid separations. It is shown that, by varying the length of the packed segment, the balance of chromatographic and electrophoretic forces can be shifted, and the selectivity can be adjusted if the separation involves the interplay of both mechanisms. Expressions are presented for estimating the retention time in a duplex column if the chromatographic and electrophoretic properties of the sample components are known. The results are expected to facilitate CEC method development in selection of the respective column segment lengths for optimum separation.

Chromatography, Liquid↗