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Biomedical subjects

D S Hage

Publications and source records attributed to D S Hage.

At least 37 records · Page 2Linked to original sources
PubMed1997

Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions.

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.

Chromatography, High Pressure Liquid↗
PubMed1997

Guidelines in selecting ligand concentrations for the determination of binding constants by affinity capillary electrophoresis.

This study examined various factors that affect the selection of ligand concentrations when using affinity capillary electrophoresis (ACE) for the determination of equilibrium constants in free solution. Two groups of model systems were used in this work: the binding of nitrophenols to alpha- or beta-cyclodextrin and the binding of D- or L-tryptophan to human serum albumin (HSA). Both systems gave 1:1 binding behavior in the ACE studies and good fits to previous equations derived to describe the shift in analyte mobility that occurs as the ligand concentration of the running buffer is varied. Some practical factors limiting the range of ligand levels that could be used in such studies included the relative amount of injected analyte, ligand solubility and the ligand's background signal. More fundamental factors included the size of the equilibrium constant for the system being investigated, the relative range of mobilities over which the analyte peak might be observed, the precision of the mobility measurements and the number of analytes present in the sample. Equations and graphs were developed for illustrating each of these latter items and their role in determining the range of ligand concentrations that could be used in ACE binding constant measurements. The results predicted by these equations and graphs showed good agreement with those observed experimentally, and should prove useful in optimizing ACE conditions for other solutes and ligands.

Cyclodextrins↗
PubMed1997

Effect of mobile phase composition on the binding kinetics of chiral solutes on a protein-based high-performance liquid chromatography column: interactions of D- and L-tryptophan with immobilized human serum albumin.

This work examined the kinetic interactions of chiral solutes on immobilized protein columns, using the binding of D- and L-tryptophan to human serum albumin as a model. Based on band-broadening studies and previous measurements of the association equilibrium constants (Ka) for this system, estimates were obtained for the dissociation and association rate constants (kd and ka) for D- and L-tryptophan under a variety of operating conditions. The relative importance of ka versus kd in creating changes in the overall binding affinity was then considered. For example, an increase in temperature from 4 to 45 degrees C gave a large change in ka for L-tryptophan that was due both to an increase in kd and to a decrease in ka, while ka and kd for D-tryptophan showed a parallel increase that led to a much smaller temperature dependence for Ka. Similar comparisons between ka, kd and Ka were performed over a range of pH values, ionic strengths and solvent polarities. It was also possible from these studies to examine the changes in enthalpy and entropy that accompanied the formation of the activated complex between human serum albumin and each solute. The results from this work were then used to illustrate the importance of kinetics and band-broadening in protein-based chiral separations, and an example was provided showing how this type of kinetic data might be used to help optimize such separations.

Chromatography, High Pressure Liquid↗
PubMed1997

Optimization of post-column chemiluminescent detection for low-molecular-mass conjugates of acridinium esters.

In this study, various factors are examined that affect the post-column chemiluminescence detection of low-molecular-mass compounds labelled with acridinium esters, using 9-phenyl acridinium ester (PAE) as the initial model. Reaction conditions examined included the effect of hydroxide or hydrogen peroxide concentration, ionic strength and surfactant content on the degree and length of light production by the acridinium ester label. Based on these results, a system was developed for the post-column detection of acridinium ester conjugates in HPLC. The final post-column reactor had optimum conditions similar to those for a benchtop luminometer but gave light production over slightly longer periods of time and had a broader range of reagent concentrations that gave a maximum response. The detection limit of this system for PAE was 7 x 10(-19) mol per injection, with linear and dynamic ranges that extended up to 3 x 10(-15) and 3 x 10(-13) mol, respectively. Some preliminary work was conducted examining the use of this system in chromatography-based competitive binding immunoassays with a thyroxine-acridinium ester conjugate being used as the label. The estimated limit of detection was 2.5 x 10(-17) mol (or roughly 10(-12) M for a 25 microliters sample) based on the retained fraction of the conjugate.

Acridines↗
PubMed1997

Determination of the diol content of chromatographic supports by capillary electrophoresis.

A capillary electrophoresis (CE) method was developed for determining the diol content of supports used in high-performance affinity or size exclusion chromatography. This method involved oxidizing the diol-bonded support with periodate, followed by the use of CE to separate and quantitate the iodate produced by this reaction. Both the oxidation and separation conditions were considered in optimizing this assay. The final method was performed by reacting a known amount of support with a 20-fold excess of periodate in pH 4.0, 0.5 M acetate buffer, with pyromellitic acid being used as an internal standard. After allowing 5-10 min for oxidation, the mixture was filtered and the filtrate was injected onto a 57 cm x 50 microns I.D. fused-silica capillary operated at 25 kV and containing pH 4.0, 0.5 M acetate as the running buffer. The total separation time was 5 min per run and gave a detection limit of 0.1 mM iodate (or 0.1 mumol diol groups) for a 6-nl injection of a 1-ml reaction mixture. By varying the amount the support that was assayed, this method could be used with either porous or non-porous supports. This technique showed good correlation with an iodometric titration but required much less sample and time to perform.

Benzoates↗
PubMed1997

Chiral separations in capillary electrophoresis using proteins as stereoselective binding agents.

Proteins are receiving increased attention in capillary electrophoresis (CE) for use as ligands in chiral separations. This review examines the types of proteins that have been employed in CE for the analysis of chiral compounds and provides a summary of applications for such an approach. Several formats for this type of analysis are discussed, including methods that use immobilized proteins (i.e., affinity capillary electrochromatography and capillary gel affinity electrophoresis) or techniques that use proteins as running buffer additives (i.e., affinity capillary electrophoresis). The role of various experimental factors in these separations are considered, and a comparison is made between the general analytical properties of CE systems that use immobilized versus solution-phase proteins.

Animals↗
PubMed1996

Chiral separation mechanisms in protein-based HPLC columns. 2. Kinetic studies of (R)- and (S)-warfarin binding to immobilized human serum albumin.

This work used plate height measurements to investigate the kinetics of (R)- and (S)-warfarin binding to an immobilized HSA column. The dissociation rate constants for (R)- and (S)-warfarin on this column increased from 0.06 to 1.9 s-1 and from 0.06 to 0.36 s-1 between 4 and 45 degrees C. The corresponding association rate constants increased from 2.4 x 10(4) to 3.2 x 10(5) M-1 s-1 for (R)-warfarin and from 4.4 x 10(4) to 7.2 x 10(4) M-1 s-1 for (S)-warfarin over the same temperature range. From the dissociation data, it was found that an increase in temperature led to a large decrease in the plate height due to stationary phase mass transfer for both enantiomers. Further studies indicated that (R)- and (S)-warfarin had similar activation energies for their binding to HSA. For (R)-warfarin, most of this energy requirement was due to the change in enthalpy of the system, while for (S)-warfarin, it was mainly due to the change in entropy. All of these results agree with an earlier model, in which (R)- and (S)-warfarin were proposed to interact with regions on the interior and exterior of HSA, respectively. In addition, these results offer a number of useful insights into the mechanisms of protein-based chiral separations.

Chromatography, High Pressure Liquid↗
PubMed1996

Role of binding capacity versus binding strength in the separation of chiral compounds on protein-based high-performance liquid chromatography columns. Interactions of D- and L-tryptophan with human serum albumin.

Frontal analysis was used to examine changes in the association constant (Ka) and moles of binding sites (mL) for D- and L-tryptophan on an immobilized HSA column under various elution conditions. Both enantiomers had single-site interactions under all conditions tested. At pH 7.0 and 25 degrees C, the strength of L-tryptophan/HSA binding was determined mostly by the change in enthalpy of the system, while D-tryptophan/HSA binding was dominated by the change in entropy. The interactions of L-tryptophan with HSA showed a large change when varying the temperature, pH, ionic strength or 1-propanol content of the mobile phase. In each case, changes in Ka accounted for most of the shifts in retention that were seen for L-tryptophan during zonal elution studies. However, mL for this compound was also affected when varying the pH and 1-propanol levels. Changes in Ka were responsible for most of the shifts in D-tryptophan retention that were seen when adjusting the mobile phase pH or ionic strength. In addition, the value of mL for D-tryptophan was affected by pH, temperature and 1-propanol levels. It was concluded that varying such chromatographic conditions can alter either the binding strength or number of binding sites for solutes injected onto immobilized protein columns.

Chromatography, High Pressure Liquid↗
PubMed1995

Studies on the rate and control of antibody oxidation by periodate.

The oxidation of antibody carbohydrate residues by periodate is a common approach for the site-specific immobilization or modification of antibodies for use in various bioanalytical methods. This study examined the time dependence of this oxidation process under a variety of pH, temperature, and concentration conditions. Polyclonal rabbit immunoglobulin G (IgG)was used as the model system for these studies. Flow-injection analysis and a hydrazide label (Lucifer yellow CH) were used to monitor the progress of the oxidation reaction. It was found that the number of oxidized sites that were available for labeling could be varied between one and eight groups per antibody by adjusting the time, pH, periodate concentration, or reaction temperature. In each case, most of these groups were produced during the first 30-60 min of the reaction. A comparison was made between these results and those of previous studies that have examined the effects of periodate treatment on amino acid residues and antibody activity. From this work, general guidelines were developed for the control and optimization of antibody oxidation for use with assays that require either high or low levels of antibody modification.

Animals↗
PubMed1995

Immunoassays.

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Animals↗
PubMed1995

Characterization of thyroxine-albumin binding using high-performance affinity chromatography. II. Comparison of the binding of thyroxine, triiodothyronines and related compounds at the warfarin and indole sites of human serum albumin.

High-performance affinity chromatography was used to examine the binding of thyroid hormones and related compounds at the warfarin and indole sites of human serum albumin (HSA). This was studied by continuously applying L-triiodothyronine (L-T3), L-reverse triiodothyronine (L-rT3) or structural analogs of these compounds to an immobilized HSA column while making injections of site-specific probe molecules (i.e. R-warfarin and L-tryptophan). The results were compared with those obtained previously for L-thyroxine (L-T4). Equilibrium association constants and thermodynamic parameters measured by this approach showed good agreement with previous models reported for L-T4 and L-T3 at their high-affinity sites on HSA. This data confirmed that the phenol groups of L-T4 and L-T3 played a significant role in the binding of these compounds at the indole site. Work performed at the warfarin site and with other solutes (e.g. L-rT3) indicated that additional factors, such as interactions through the thyronine backbone or terminal amine and carboxyl groups of these compounds, could also be involved in the binding of thyroid hormones to HSA.

Binding Sites↗
PubMed1995

Characterization of the protein binding of chiral drugs by high-performance affinity chromatography. Interactions of R- and S-ibuprofen with human serum albumin.

Zonal elution and high-performance affinity chromatography were used to study the different binding characteristics of R- and S-ibuprofen with the protein human serum albumin (HSA). This was done by injecting small amounts of R- and S-ibuprofen onto an immobilized HSA column in the presence of a mobile phase that contained a known concentration of R- or S-ibuprofen as a competing agent. These studies indicated that R- and S-ibuprofen had one common binding site on the immobilized HSA column. In addition, S-ibuprofen had at least one other major binding region. The association equilibrium constant for R-ibuprofen with HSA was found to be 5.3 x 10(5) M-1 at pH 6.9 and 25 degrees C. Under the same conditions, the association constants for S-ibuprofen at its two sites were 1.1 x 10(5) M-1 and 1.2 x 10(5) M-1. The S-ibuprofen sites were present in about a 1:1 ratio and appeared to exhibit some allosteric interactions at high S-ibuprofen concentrations. The chromatographic technique used in this work is a general one which can be adapted for use in studying the interactions of other chiral compounds with either HSA or additional proteins.

Binding, Competitive↗
PubMed1995

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection.

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with beta-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts were injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C18 and C8 reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 micrograms/l for 20-microliters injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 micrograms/l total MHPG was +/- 6% and the day-to-day precision was +/- 15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.

Acetates↗
PubMed1994

Determination of urinary albumin using high-performance immunoaffinity chromatography and flow injection analysis.

The detection of low levels of albumin in urine can be an important aid in the early diagnosis of kidney disease. In this study, an automated system for urine analysis was developed that could simultaneously measure both albumin and creatinine, an indicator of urine output and volume. Albumin was determined by use of a column that contained immobilized anti-albumin antibodies. An on-line flow injection analysis system was used to measure creatinine as this solute eluted nonretained from the antibody column. The total analysis time of the system was 5 min per injection. Limits of detection for albumin and creatinine in a 20 microL sample (at S/N = 2) were 3 and 5 mg/L, respectively. The response for these compounds covered the range of clinical interest. Within-run precision for both analytes was +/- 1-2% at normal urine concentrations. The results of this method showed good agreement with those obtained by reference techniques. This approach can be adapted for the detection of other urine components by changing the type of antibody column used in the system.

Albuminuria↗
PubMed1994

Chiral separation mechanisms in protein-based HPLC columns. 1. Thermodynamic studies of (R)- and (S)-warfarin binding to immobilized human serum albumin.

This work characterizes the thermodynamic processes involved in the binding and separation of (R)- and (S)-warfarin on a high-performance human serum albumin (HSA) column. Frontal analysis was used to determine the strength and degree of binding for each enantiomer. (R)- and (S)-warfarin were found to bind at the same region on HSA; however, (R)-warfarin had a larger number of column binding sites. The number of binding sites for both enantiomers showed a slight increase with temperature. The total changes in free energy for (R)- and (S)-warfarin binding were similar at 37 degrees C, but the contribution due to entropy was greater for the R-enantiomer. These results suggested that (R)-warfarin was interacting mainly with the binding site interior, while (S)-warfarin interacted more with the site's outer surface. This model was confirmed by examining the retention of (R)- and (S)-warfarin on the HSA column under various pH, ionic strength, and organic modifier conditions. The different changes in entropy for these solutes made it possible to vary their separation by changing column temperature. Both thermodynamic properties and column binding capacities were found to be important in determining the degree of separation obtained for these compounds.

Chromatography, High Pressure Liquid↗
PubMed1994

Chiral separations in capillary electrophoresis using human serum albumin as a buffer additive.

This study examined the theory and mechanisms of chiral separations in capillary electrophoresis based on the use of proteins as buffer additives. Human serum albumin (HSA) was used as the model ligand; D,L-tryptophan and (R,S)-warfarin were used as the test analytes to be separated by this protein. Items examined in this work included the amount of HSA adsorbed to the capillary wall and the stability of this adsorbed protein layer. These were investigated by performing frontal analysis on the capillary with HSA and by injecting neutral markers through the capillary at different applied voltages before and after HSA treatment. The role of adsorbed HSA vs HSA in the buffer in determining the stereoselectivity of the CE system was also examined. Adsorbed HSA was the predominant agent involved in the separation of (R,S)-warfarin, while HSA in the buffer had the most significant effect in the resolution of D,L-tryptophan. Two distinct separation mechanisms were proposed to explain these differences. Good agreement was obtained between the results predicted by these mechanisms and the experimental data. Under optimized conditions, both pairs of enantiomers were separated with baseline resolution in less than 12 min.

Adsorption↗
PubMed1994

Development of dihydrazide-activated silica supports for high-performance affinity chromatography.

A method of preparing dihydrazide-activated silica was developed for use in high-performance affinity chromatography (HPAC). This support was made by oxidizing diol-bonded silica and reacting it with oxalic or adipic dihydrazide. The steps involved in this synthesis were studied and confirmed by FTIR. Items considered in optimizing the preparation of the support included the amount of dihydrazide added and the reaction time or pH used. Control of dihydrazide bifunctional attachment was obtained by varying the extent of diol-bonded silica oxidation. This support was successfully used in the immobilization of oxidized antibodies, horse radish peroxidase (a glycoenzyme) and transfer RNA. In each case, data indicated that immobilization was through site specific coupling rather than non-specific adsorption. Dihydrazide-activated silica was found to be stable for 2-6 weeks after preparation when stored at 5 to 25 degrees C. The linkage between oxidized biomolecules and this support was stable for at least one month in the presence of various solvents commonly used in HPAC.

Adipates↗