Biomedical subjects

C Horváth

Publications and source records attributed to C Horváth.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1998

Axial nonuniformities and flow in columns for capillary electrochromatography.

Unlike in capillary zone electrophoresis and microscale high-performance liquid chromatography, columns in capillary electrochromatography (CEC) have discontinuities of the electric field strength and the flow velocity at the interface of the packed and open segments of the column. The goal of the present work is to offer a framework for measuring and interpreting the relevant electrochromatographic parameters such as the electric field strength, the potential drop, and the flow velocity in the packed segment of a CEC column. This would help us gain further insight in the electrochromatographic process and facilitate the design of CEC separation technology and comparison of data from different sources. First, the flow of ions that is governed by the conservation of current is analyzed and the potential drop across the packed and open segments of the column calculated. Then, conservation of volumetric flow rate is used to calculate the flow velocities through the two segments and, further, to estimate the net flow velocity through such a column. To satisfy the mass conservation law, in most cases a "flow-equalizing intersegmental pressure", which is different from the pressures at the two ends of the column, develops at the interface of the packed and the open segments. The intersegmental pressure, induced to equalize the flow rates in the two segments, has been shown to have a significant effect on the magnitude as well as the radial distribution of the flow velocity in the open segment, where the net flow becomes a mixture of electroosmotic and pressure-driven flows.

Chromatography, High Pressure Liquid↗

PubMed1998

Sintered octadecylsilica as monolithic column packing in capillary electrochromatography and micro high-performance liquid chromatography.

Fused-silica capillaries were packed with porous 6-microns octadecylated silica microspheres and subjected to thermal treatment in order to obtain a column with porous silica based monolithic packing. After sintering, the monolithic packing was reoctadecylated in situ with dimethyloctadecylchlorosilane. The mechanical strength and stability of the monolithic column were significantly greater than those of conventional columns packed with particulate stationary phase. The performance of the columns was evaluated in both mu-HPLC and capillary electrochromatography (CEC) of small aromatic compounds and polycyclic aromatic hydrocarbons with 10 mM borate, pH 8.0, in acetonitrile-water mixtures as the mobile phase. Since no untoward bubble formation was observed, CEC could be performed in a CZE unit without the need for pressurizing the monolithic column. The plate efficiency of the monolithic column was similar to that of columns freshly packed with the same particles and was always higher in the CEC than in the mu-HPLC under otherwise identical conditions. The electrosmotic flow (EOF) velocity increased with the acetonitrile content of the eluent in open fused-silica capillaries with octadecylated innerwall and in columns packed with sintered octadecylsilica. In contradistinction, the EOF velocity decreased with the organic strength of the eluent in raw fused-silica capillaries. The opposite trend is attributed to the different effect of changing organic modifier concentration on the accessibility of silanol groups at the raw and octadecylated silica surfaces.

Chromatography, High Pressure Liquid↗

PubMed1998

Cyclodextrin aided separation of peptides and proteins by capillary zone electrophoresis.

Carboxymethylated-beta-cyclodextrin (CMBCD) in the electrophoretic medium (aqueous 50 mM sodium phosphate, pH 2.5) enhanced the separation using raw fused-silica capillaries in CZE of the four standard proteins: alpha-chymotrypsinogen A, cytochrome c, lysozyme and ribonuclease A. Furthermore, with 20 mM CMBCD in the electrophoretic medium, the cis-trans isomers of angiotensin could be separated at room temperature, whereas the separation of the conformers required subambient temperatures as low as -20 degrees C without CMBCD in the electrophoretic medium [50 mM sodium phosphate (pH 2.5), containing 10% (v/v) methanol]. Addition of heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMBCD) had no effect on the separation of the above proteins and peptides. The results suggest that in microcolumn separation techniques, certain cyclodextrin additives can be useful selectivity enhancers.

Amino Acid Sequence↗

PubMed1998

Cyclodextrins as selectivity enhancers in capillary zone electrophoresis of proteins.

The selectivity in the capillary zone electrophoresis (CZE) of a variety of acidic and basic proteins including alpha-chymotrypsinogen A, cytochrome c, lysozyme, ribonuclease A, ovalbumin, and beta-lactoglobulins A and B, was altered by adding 6-monodeoxy-6-monoamino-beta-cyclodextrin or carboxymethylated beta-cyclodextrin to the electrophoretic medium of aqueous 50 mM sodium phosphate, pH 2.5. On the other hand, no significant improvement was obtained in the separation upon addition of heptakis (2,6-di-O-methyl)-beta-cyclodextrin. Whereas protein adsorption on the wall of raw silica capillaries was significant in the absence of cyclodextrin, by addition of beta-cyclodextrin or its derivatives to the background electrolyte, wall adsorption was reduced with concomitant enhancement of the recovery. The results confirm that in various separation techniques, particularly those which employ microcolumns, certain cyclodextrin additives can be useful selectivity enhancers not only in the separation of small sample molecules but also in that of proteins.

Adsorption↗

PubMed1998

Sodium transport and bone mineral density in hypercalciuria with thiazide treatment.

Erythrocyte sodium-potassium (Na+/K+)-ATPase and sodium-lithium (Na+/Li+) countertransport activities were measured in 18 children (aged 9.6 years, range 6-16 years) with idiopathic hypercalciuria (IHU) to evaluate cellular Na handling. The effect of chronic thiazide administration on these parameters and on bone mineral density was also evaluated. Patients with IHU had significantly lower erythrocyte Na+/K+-ATPase activity than 23 age-matched healthy controls (mean +/- SEM 2,156 +/- 110 micromol P/l erythrocyte per hour vs. 3,165 +/- 175, P < 0.01). Thiazide treatment significantly lowered urinary calcium excretion; this was followed by a slight suppression of intact parathyroid hormone (iPTH). The urinary calcium/creatinine ratio before and during treatment was 0.90 +/- 0.07 mmol/mmol versus 0.51 +/- 0.06 respectively, P < 0.01. The corresponding iPTH levels were 5.9 +/- 0.6 pmol/l and 5.1 +/- 0.7, P < 0.05. The Na+/K+-ATPase activity increased significantly (2,769 +/- 169 micromol P/l erythrocyte per hour vs. 2,156 +/- 110 in the control period, P < 0.01) and the Na+/Li+ countertransport decreased (268 +/- 28 micromol Li/l erythrocyte per hour vs. 328+26 in the control period, P < 0.03). The bone mineral density Z score rose from -1.3 +/- 0.26 to -0.8 +/- 0.22 (P < 0.03). We conclude that IHU is accompanied by abnormalities of erythrocyte Na+/K+-ATPase and Na+/Li+ countertransport which are corrected by chronic hydrochlorothiazide administration. These changes could model alterations in renal tubular transport mechanisms still to be elucidated. Chronic thiazide treatment also has a positive effect on bone mineral density.

Adolescent↗

PubMed1998

Effect of neonatal glucocorticoid treatment on bone mineralization of adult nontreated, dexamethasone-treated or vitamin D3-treated rats.

1. Single neonatal dexamethasone (DEX) treatment significantly decreased the body weights of 5-month old male rats. There was no significant difference in females. 2. Bone mineral density (BMD) of neonatally DEX-treated male rats and bone mineral content (BMC) of double DEX-treated (neonatally and in adult age) males were reduced. 3. BMD and BMC calculated to body weight were highly significantly increased after neonatal or double DEX treatment in males. In females only BMC/body weight was elevated after double DEX treatment. 4. Adult vitamin D treatment completely compensated for the changes caused by single or double DEX treatment. 5. The results call attention to the imprinting effects of neonatal glucocorticoid treatment, which were manifested in changes of body weight and bone mineral mass. At the same time the gender-dependence of this phenomenon was demonstrated.

Aging↗

PubMed1998

Effect of single neonatal vitamin D3 treatment (hormonal imprinting) on the bone mineralization of adult non-treated and dexamethasone treated rats.

Hormonal imprinting (the first encounter between the hormone and receptor after birth) is needed for the normal development of receptor. Presence of the appropriate hormone in excess, or its absence, as well as presence of hormone-like molecules able to bind to the maturing receptor in this time, can cause faulty imprinting. In this experiment the effect of neonatal treatment with a single dose of 0.05 mg cholecalciferol (vitamin D3) was studied by bone densitometry. The treatment caused significant decrease of body weight in 3-month old females and also significant reduction of bone mineral density (BMD) and bone mineral content (BMC) in males. Dexamethasone treatment of 3-month old rats for 10 days increased BMD in males and BMC in females without affecting body weight. The double treatment (vitamin D neonatally and dexamethasone when adult) decreased the body weight of both sexes and increased BMD in males, and BMC, BMD/bw and BMC/bw in both sexes, related to the control or the only vitamin D treated groups. Considering the hormonal imprinting effect of neonatal vitamin D treatment at glucocorticoid receptorial level in other experiments, similar effects also can be supposed for vitamin D itself, manifested in the changes of bone mineralization.

Animals↗

PubMed1997

A biophysical study of integral membrane protein folding.

In order to characterize the thermodynamic constraints on the process of integral membrane protein folding and assembly, we have conducted a biophysical dissection of the structure of bacteriorhodopsin (BR), a prototypical alpha-helical integral membrane protein. Seven polypeptides were synthesized, corresponding to each of the seven transmembrane alpha-helices in BR, and the structure of each individual polypeptide was characterized in reconstituted phospholipid vesicles. Five of the seven polypeptides form stable transmembrane alpha-helices in isolation from the remainder of the tertiary structure of BR. However, using our reconstitution protocols, the polypeptide corresponding to the F helix in BR does not form any stable secondary structure in reconstituted vesicles, and the polypeptide corresponding to the G helix forms a hyperstable beta-sheet structure with its strands oriented perpendicular to the plane of the membrane. [The polypeptide corresponding to the C helix spontaneously equilibrates in a pH-dependent manner between a transmembrane alpha-helical conformation, a peripherally bound nonhelical conformation, and a fully water soluble conformation; the conformational properties of this polypeptide are the subject of the accompanying paper: Hunt et al. (1997) Biochemistry 36, 15177-15192.] Our observations suggest that the folding of alpha-helical integral membrane proteins may proceed spontaneously. However, the preference for a non-native conformation exhibited by two of the polypeptides suggests that the formation of some transmembrane substructures could require external constraints such as the links between the helices, interactions with the rest of the protein, or the involvement of cellular chaperones or translocases. Our results also suggest a strategy for improving the thermodynamic stability of alpha-helical integral membrane proteins, a goal that could facilitate attempts to overexpress and/or refold them.

Amides↗

PubMed1997

Capillary zone electrophoresis with fluid-impervious polymer tubing inside a fused-silica capillary.

A fused-silica capillary was used as a mold in the in situ formation of, and as a sheath for, a highly crosslinked poly(styrene-divinylbenzene) inner tubing having 3-5 microns wall thickness. This 'tube-in-the-tube' construction with the thin-walled, fluid-impervious polymer inner tubing precluded contact between the aqueous buffer solution and the inner wall of the fused-silica capillary. As a result, this structure withstood long-term treatment with highly alkaline solutions without deterioration. In order to hydrophilize the inner surface of the polymer tube, a polyoxyethylene oligomer was grafted to its inner wall and subsequently crosslinked. The inner tube with such hydrophilic coating was also stable to hydrolytic attack by 1 M NaOH. Although the nonpolar polymeric inner surface generated electrosmotic flow as if it had some fixed negative charges, the flow velocity became almost negligibly small once it had been hydrophilized. As illustrated by electropherograms, the hydrophilization of the polymeric inner tube greatly facilitated the CZE of basic proteins in the pH range from 3 to 6 without the need for additives in the electrophoretic medium to mask the silanol groups at the surface of quartz capillaries.

Chemical Phenomena↗

PubMed1997

Temperature programming and gradient elution in reversed-phase chromatography with packed capillary columns.

The two major anisocratic elution modes were compared in reversed-phase chromatography with 180 microns I.D. fused-silica capillary columns packed with 6 microns Zorbax SB ODS-silica. By evaluating the retention factors of alkylbenzenes at acetonitrile concentrations varying from 60 to 80% (v/v) in the aqueous eluent and in the temperature range of 30-80 degrees C, it was found that a 5 degrees C change in column temperature and a 1% change in acetonitrile concentration have almost the same effect on retention. This is illustrated by the almost identical chromatograms of an alkylbenzene sample obtained by temperature programming and by gradient elution under the same conditions otherwise and by simulation of the trajectories of the eluent peaks moving down the column. The results suggest that in reversed-phase HPLC with packed capillary columns temperature programming offers an alternative to gradient elution in a relatively narrow range of the required elution strength. Thermodynamic data from isocratic chromatographic measurements were used to predict the retention times of alkylbenzenes in reversed-phase chromatography with temperature programming at different heating rates and column inlet pressures. Temperature programming was used to separate beta-lactoglobulins A and B by reversed-phase chromatography. It was also employed concomitantly with gradient elution to enhance the separation of a mixture of four standard proteins. The results indicate that temperature programming could serve as an adjunct to gradient elution by means of fine retention tuning to bring about or increase the resolution of closely related macromolecules.

Chromatography, High Pressure Liquid↗

PubMed1997

Displacement chromatography with on-column isomerization.

Displacement chromatography was simulated for the separation of two feed components interconverting by a reversible first order reaction and with Langmuirian adsorption behavior. The study was prompted by recent interest in the isolation of cis and trans forms of peptides containing one or more peptidyl-proline residues when the isomerization reaction interferes with the separation. The parameter values used in the simulations are similar to those found experimentally by reversed-phase chromatography and capillary electrophoresis of phenylalanine-proline dipeptide. From the concentration profiles computed by the finite difference scheme, the dependence of both the yield and production rate on the temperature, column length, flow velocity and displacer concentration was evaluated. The most important operational variable of the system is the temperature as it affects both the kinetic and adsorption parameters. The yield and production rate of the component of interest were evaluated as a function of the column length and displacer concentration under conditions that facilitate its efficient separation and the plots show an optimum. Nonetheless, optimal conditions for yield and production rate were considerably different. In the temperature range from 2 to 42 degrees C, the yield always decreases with increasing temperatures and for all the cases, optimum yield by displacement mandates the use of conditions such as pH, solvent and temperature under which the rate of interconversion is reduced to a level where it does not palpably interfere with the separation. On the other hand, under certain conditions optimal production rate can be obtained at higher temperatures.

Chromatography, High Pressure Liquid↗

PubMed1997

Capillary electrochromatography with gradient elution.

A capillary electrochromatograph incorporating a gradient-forming system generally employed in HPLC is described, and the use of gradient elution in reversed phase electrochromatography is demonstrated by the separation of PTH-amino acids and steroid hormones. The gradient former employs two reciprocating displacement pumps to control the composition of the eluent in the reservoir at the column inlet with time in a controlled manner. Thus, the composition of the mobile phase flowing through the column and driven by electrosmotic forces can be changed with time in a controlled fashion as customary in HPLC with gradient elution. The design of the system allows also for isocratic elution by pumping the eluent of constant composition through the cavity at the column inlet and thus continuously supplying fresh buffer. The eluent gradient is generated by the two pumps and a 10 microL mixer. From there the liquid passes at a flow rate of 0.1-0.2 mL/min through the 17 microL cavity housing the column inlet and an electrode. The flow of the mobile phase was electrosmotic at an effective overall electric field strength of 500-1500 V/cm through a 50 microns x 20/12 cm capillary column packed with 3.5 microns octadecylated silica particles. Gradient profiles generated in this manner were highly reproducible. The same-day and day-today reproducibilities of the electrosmotic flow were found to be better than 3%. The use of the capillary electrochromatographic system was demonstrated with isocratic and gradient elution for the separation of complex mixtures of biologically interesting substances. The influence of the column temperature on the electrosmotic flow velocity and retention of PTH-amino acids was also investigated.

Chromatography, High Pressure Liquid↗

PubMed1997

Dynamics of capillary electrochromatography experimental study on the electrosmotic flow and conductance in open and packed capillaries.

For capillary electrochromatography (CEC) to become an analytical separation technique of high speed and resolution the factors determining the conductivity of the column as well as the generation and control of electrosmotic flow (EOF) in porous media have to be understood. In the present study the conductance of capillaries packed with a variety of stationary phases was evaluated with respect to the conductance of the open capillary and the data were interpreted in the light of the Tobias equation. However, the consistently observed reduction of the EOF when a capillary having a charged inner wall is packed with particles having charges of the same sign and the dependence of the EOF velocity on the particle size needs further explanation. The data suggests that, due to the employment of relatively long columns packed with small particles, CEC may offer peak capacities much higher than HPLC or micro-HPLC. The CEC columns are unique as they consist of a packed and an open capillary segment having different conductances and consequently different voltage gradients and electrical field strengths. Therefore, any sufficiently detailed study on CEC systems requires also the characterization of the individual column segments. EOF velocities of 6-7 mm/s could be realized at 60 kV applied voltage with a 23/32 cm x 50 microns raw fused-silica capillary packed with 6-micron Zorbax ODS particles. The current was a linear function of the field strength up to 1.8 kV/cm, but at high field strengths the EOF increased with squared field strength. Data on band spreading indicate that with a given column the plate height at high EOF velocities is smaller in CEC than in micro-HPLC and it is weakly dependent on the velocity.

Acetonitriles↗

PubMed1997

Capillary electrochromatography: theories on electroosmotic flow in porous media.

In view of the present interest in capillary electrochromatography (CEC), theories dealing with electroosmotic flow (EOF) in porous media are reviewed with particular regard to the use of packed capillaries in CEC. Two of the models found in the pertinent literature are applicable to CEC and give simple analytical solutions. The first of the two models is based on von Smoluchowski's work as adapted and extended by Overbeek. It deals with EOF through packed capillaries under conditions of low electric field strength where the EOF varies linearly with the field strength because there is no polarization of the double layer. Overbeek's model originally developed for porous media of infinite dimensions was modified in an attempt to account for the wall effect that assumes importance in the packed capillary columns used in CEC. The second model proposed by Dukhin and his coworkers predicts EOF of at least an order of magnitude higher than that expected by classical theories. This "electroosmosis of the second kind" is believed to occur in columns packed with conductive particles like ion exchangers at high electric field strengths when the double layer is polarized and the EOF becomes a non-linear function of the applied voltage. Conditions necessary for electroosmosis of the the second kind are likely to arise upon the further development of CEC when further enhancement of the speed of analysis is brought about at electric field strength higher than that employed at present.

Electric Conductivity↗

PubMed1997

[Urinary calcium excretion. (Normal values for urinary calcium/creatinine ratio in Hungary). Multicenter study].

Metabolic bone disorders have attracted increasing attention in Hungary due to their significant impact on public health care. Measuring urinary calcium excretion is the first step in the biochemical assessment of bone metabolism. Fasting urinary calcium corrected by creatinine excretion is widely used all over the world. The aim of the present study was to establish standard methods and normal values for the calcium/creatinine ratio in Hungary. Twenty-four centers specializing in metabolic bone diseases participated in the study. Urine standards were sent out to these centers for calcium and creatine determinations. Based on the collected data, methods were corrected in order to achieve similar results for the standards. In the second phase of the study, the normal values for calcium/creatinine ratio were determined in SI units based on the data from 1846 healthy subjects (age 20-80 yrs) including 944 females and 902 males. The normal value for females was 0.438 +/- 0.391 (means +/- SD), and 0.395 +/- 0.352 for males, respectively (p < 0.03). The ratio increased with age in both sexes. The highest values were observed between 60-64 years in women and 70-74 years in men, respectively. After this peak, the calcium/creatinine ratio decreased. The values before and after 45 years of age were significantly different both in women (0.37 +/- 0.36, vs. 0.52 +/- 0.41, p < 0.001; and men (0.32 +/- 0.23, vs. 0.47 +/- 0.45, p < 0.001). The use of this distinction is recommended in the everyday practice.

Adult↗

PubMed1997

Capillary zone electrophoresis at subzero temperatures II: chiral separation of biogenic amines.

The separation of enantiomer pairs of eight biogenic amines by capillary zone electrophoresis (CZE) was investigated with heptakis (2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector at temperatures ranging from -20 degrees to 40 degrees C by using a commercial electrophoresis unit retrofitted with an external thermostated refrigerated circulating bath in order to assist the original cooling system. Sodium phosphate in both neat aqueous and methanolic media at pH 2.5, as measured by the glass pH electrode, were used with the fused silica capillary from 1.5 degrees to 40 degrees C and -20 degrees to 40 degrees C, respectively. The effect of temperature on enantioselectivity was found to depend on the number of phenolic hydroxyl groups in the molecule. Upon lowering the temperature from 40 degrees to -20 degrees C, the chiral selectivity of the system, as measured by the relative mobility difference, increased tenfold for the amines with two vicinal phenolic hydroxyls, whereas the increase was insignificant for those having no phenolic hydroxyl groups. The complex formation constants of three amines which have the same molecular structure but the number of phenolic hydroxyl groups were determined at different temperatures and the thermodynamic parameters as well as compensation temperatures for the process were evaluated. Whereas the compensation temperature was 690 K for the amine without phenolic hydroxyl group, it was < 400 K for the amines with one or two phenolic hydroxyl groups. The difference in the compensation temperatures indicates that the intrinsic mechanisms of their complexation with the chiral selector are not the same; this may account for the discrepancies observed in the temperature dependency of the chiral selectivity. The enthalpy change per phenolic hydroxyl group was 2.5 kcal mol(-1), which compares favorably with the typical value for a single hydrogen bond. Therefore, when the amines have phenolic hydroxyl groups, the strong increase in chiral selectivity with decreasing temperature may be due to enhanced H-bonding between the cyclodextrin and the phenolic hydroxyls under the conditions employed in this study.

Biogenic Amines↗