PubMed Health⌕ Search

Biomedical subjects

B Loun

Publications and source records attributed to B Loun.

10 recordsLinked to original sources
PubMed1997

Effects of ligand heterogeneity in the characterization of affinity columns by frontal analysis.

This study examined how the binding capacities and equilibrium constants measured by frontal analysis are affected by ligand heterogeneity in affinity columns. Equations derived for n- and two-site systems gave good agreement with results obtained for the binding of L-thyroxine to a column containing human serum albumin (HSA) and for the binding of (R)-warfarin to coupled columns containing HSA or pigeon serum albumin. The same equations were used to examine how different degrees of ligand heterogeneity affected the apparent binding capacities or equilibrium constants measured using the linear range of double-reciprocal frontal analysis plots. A large proportion of two-site systems gave good estimates (i.e., less than 10-20% error) for the true total column capacity and for the association constant of the highest affinity ligand in the column. A smaller, but still appreciable, fraction of all three- and four-site cases also produced good estimates of these values. The results of this work are not limited to protein-based affinity columns but should be applicable to any type of stationary phase that has well-defined binding regions and relatively fast, reversible interactions with solutes.

Chromatography, Affinity↗
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

Adaptation of a quantitative immunoassay for urine myoglobin. Predictor in detecting renal dysfunction.

Myoglobinuria, subsequent to rhabdomyolysis, may cause acute renal failure. For this reason, many qualitative and quantitative tests have been developed for the detection of myoglobin in urine. The authors describe the adaptation and optimization of the Stratus II serum myoglobin immunoassay to quantify urine myoglobin. In addition, the assay was used to accurately determine urine myoglobin concentrations in subjects at potential risk for myoglobin-induced renal dysfunction and the results obtained compared to conventional qualitative methods for urine myoglobin. The assay demonstrated with-in run and between-run coefficient of variations (CVs) of 6.2% and 7.2%, respectively, was linear from 0-950 micrograms/L, demonstrated good recovery, and was free from interference by hemoglobin, creatinine, and urea. Specimens were diluted with 0.1 mol/L phosphate buffer, pH 9.0 containing 3% bovine serum albumin before analysis. Myoglobin was assayed on urine obtained from 30 patients suspected of having myoglobinuria. Fifteen of 17 patients with serum creatinine greater than 1.4 mg/dL had myoglobin concentrations greater than 20,000 micrograms/L, whereas the remaining 31 patients with normal serum creatinine had urine myoglobin concentrations of less than 18,000 micrograms/L. If serum creatinine is used as an indicator of renal function, it would appear that accurate measurement of urine myoglobin may facilitate identification of patients with increased susceptibility to myoglobin-induced acute renal failure.

Acute Kidney Injury↗
PubMed1996

Ultrafiltration discrepancies in recovery of myoglobin from urine.

We investigated the efficiency, accuracy, and reliability of the ultrafiltration/dipstick methodology commonly used to diagnose myoglobinuria. Twenty-five myoglobin-containing urine specimens were filtered by centrifugation for 15 min at 1500g through a Centricon-30 membrane filter. Both the original specimen and filtrate were assayed for myoglobin. The amount of myoglobin recovered subsequent to filtration varied from <1-38%. This poor and variable recovery was independent of sample matrix or precentrifugation of the specimens. This was most critical for urine specimens with myoglobin concentrations <60 000 microg/L. Fourteen of 18 such filtrates had concentrations <350 microg/L, a concentration below which a negative result would be obtained by using conventional dipstick methods. Thus, the use of this procedure has the potential to misdiagnose patients with myoglobin concentrations associated with increased risk of subsequent renal dysfunction, in particular when urine myoglobin concentrations are <60 000 microg/L.

False Negative Reactions↗
PubMed1996

Pneumatic transport exacerbates interference of room air contamination in blood gas samples.

OBJECTIVE: To characterize and control the potential interference to P(O2) determinations when blood contaminated with air is sent via a pneumatic tube system (PTS). DESIGN: Both tonometered blood at P(O2)s of 65, 75, 142, and 339 mm Hg and arterial blood gas samples from patients with baseline P(O2)s from 70 to 400 mm Hg were analyzed for P(O2) to determine possible effects of air contamination from PTS transport. SETTING: A large teaching hospital in which a variety of personnel routinely send samples to the laboratory by PTS transport. PATIENTS: Twenty patients under anesthesia for elective surgery and 21 patients in an intensive care unit who had a wide range of P(O2)s. Several additional patients with a preexisting lung pathology likely to cause hypoxemia were selected to provide samples with low P(O2)s. MAIN OUTCOME MEASURES: Measurement of bias in P(O2) between samples sent via PTS and samples walked to the laboratory. RESULTS: Interference from air contamination was worse after PTS transport compared with manual transport of the specimen. Over a wide range, the P(O2) in specimens after PTS transport tended toward 160 mm Hg. Samples from hypoxemic patients were prone to errors in P(O2) that could have resulted in clinical misinterpretation; 5 of 10 samples with a baseline P(O2) less than 85 mm Hg had increases of 10 mm Hg or more when contaminated with air. Cooling samples with high P(O2)s minimized changes to P(O2), probably by increasing the solubility of oxygen. Mechanical buffering by various liners used in the carriers did little to alleviate the interference. Decreasing the speed of pneumatic transport by 50% lessened the effect on P(O2). CONCLUSION: Interference can be minimized by carefully purging samples of all air bubbles using the following protocol: invert syringe to check for air bubbles, then retap and reexpel bubbles if necessary. Personnel that collect and send blood gas samples via PTS should be educated about the problem of interference. Modifications both to pneumatic sample transport systems and to blood gas syringes should be investigated to minimize the effect.

Air↗
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

Intracellular magnesium content of mononuclear blood cells and granulocytes isolated from leukemic, infected, and granulocyte colony-stimulating factor-treated patients.

The intracellular magnesium (Mg) concentration of granulocytes and mononuclear blood cells (MBCs) was determined in cells isolated from patients with several disorders. The mean (+/-SD) Mg content of MBCs isolated from patients diagnosed with lymphocytic leukemia, myelocytic leukemia, or infection; from patients treated with granulocyte colony-stimulating factor (G-CSF); and from healthy volunteers (control group) was 2.3 (+/-0.6), 3.3 (+/-0.5), 4.1 (+/-0.8), 3.9 (+/-0.4), and 3.9 (+/-0.6) fmol/cell, respectively. The Mg content of MBCs isolated from patients with lymphocytic and myelocytic leukemia, but not those from patients with infection or receiving G-CSF treatment, were significantly lower (P < 0.001) than those from the control subjects. The mean Mg concentration of granulocytes obtained from lymphocytic leukemia, myelocytic leukemia, infection, and G-CSF patients and from the control group was 3.2 (+/-0.9), 3.4 (+/-0.5), 3.8 (+/-0.6), 4.5 (+/-0.6), and 4.6 (+/-0.6) fmol/cell, respectively. Granulocytes isolated from leukemic and infectious patients yielded lower intracellular Mg concentrations (P < 0.005) than those from patients receiving G-CSF and the control group. This study demonstrates that intracellular Mg content is altered in several pathological states. Several factors, including depleted Mg stores or altered intracellular Mg binding sites, could be responsible for these changes. Apparently, intracellular Mg content may be of use in assessing total body Mg status.

Granulocyte Colony-Stimulating Factor↗
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↗
PubMed1992

Characterization of thyroxine-albumin binding using high-performance affinity chromatography. I. Interactions at the warfarin and indole sites of albumin.

A high-performance affinity column containing immobilized human serum albumin (HSA) was used to study the binding of thyroxine at the warfarin and indole sites of HSA. Frontal analysis, using R-warfarin and L-tryptophan as probes for these sites, demonstrated that the immobilized HSA had binding behavior equivalent to that observed for HSA in solution. By injecting R-warfarin or L-tryptophan in the presence of excess thyroxine, it was found that thyroxine was binding directly to both types of site. The warfarin and indole sites had relatively strong binding for thyroxine, with association constants at 37 degrees C of 1.4 x 10(5) and 5.7 x 10(5) M-1, respectively. The value of delta G for these sites ranged from -7 to -8 kcal/mol and had a significant entropy component. The techniques used in this study are not limited to thyroxine-HSA interactions, but should also be valuable in examining the site-specific binding of other drugs and hormones to HSA.

Chromatography, Affinity↗