Luminance and color contrast sensitivity and VEP latency in subjects with normal and defective binocularity

The effect of an additive (Brij 35) on the mobilities of a group of porphyrin acids is quantitatively characterized based on a 1:1 dynamic complexation model. Varying additive concentration shifts the equilibrium and changes the viscosity of the background electrolyte. The equilibrium constant, the electrophoretic mobility of the free analyte, and the electrophoretic mobility of the complex are identified as the parameters necessary to describe the analytes' migration behavior. Several statistical methods for obtaining these parameters are discussed. The equilibrium constants and complex mobilities are calculated using three different linear regression methods. The weighted y-reciprocal method was preferred because it gives smaller error, and the data points are evenly distributed along the concentration axis. These values are confirmed using a nonlinear regression to ensure that the proper weighting was used in the linear regression plots. The parameters are then used to predict the apparent mobilities of the analytes over the entire additive concentration range, allowing the optimum separation conditions to be identified. For disc-like molecules, such as porphyrins, the mobility is determined by the orientation of the molecule in an electric field, in addition to their size and charge. The strength of binding between the porphyrins and Brij 35 depends on the number of binding sites and the solvation shell.     

The development of a new method to detect the adulteration of commercial aloe gel powders

Recent high-resolution analysis of tubulin's structure has led to the prediction that the taxol binding site and a tubulin acetylation site are on the interior of microtubules, suggesting that diffusion inside microtubules is potentially a biologically and clinically important process. To assess the rates of transport inside microtubules, predictions of diffusion time scales and concentration profiles were made using a model for diffusion with parameters estimated from experiments reported in the literature. Three specific cases were considered: 1) diffusion of alpha beta-tubulin dimer, 2) diffusion/binding of taxol, and 3) diffusion/binding of an antibody specific for an epitope on the microtubule's interior surface. In the first case tubulin is predicted to require only approximately 1 min to reach half the equilibrium concentration in the center of a 40 microns microtubule open at both ends. This relatively rapid transport occurs because of a lack of appreciable affinity between tubulin and the microtubule inner surface and occurs in spite of a three-fold reduction in diffusivity due to hindrance. By contrast the transport of taxol is much slower, requiring days (at nM concentrations) to reach half the equilibrium concentration in the center of a 40 microns microtubule having both ends open. This slow transport is the result of fast, reversible taxol binding to the microtubule's interior surface and the large capacity for taxol (approximately 12 mM based on interior volume of the microtubule). An antibody directed toward an epitope in the microtubule's interior is predicted to require years to approach equilibrium. These results are difficult to reconcile with previous experimental results where substantial taxol and antibody binding is achieved in minutes, suggesting that these binding sites are on the microtubule exterior. The slow transport rates also suggest that microtubules might be able to serve as vehicles for controlled-release of drugs.     

Study of the effective dose of a topical antifungal agent, omoconazole nitrate, on the basis of percutaneous pharmacokinetics in guinea-pigs and mice

The clinically useful optimum dose of omoconazole nitrate, a topical antifungal agent, has been examined by analysing the percutaneous pharmacokinetics of the drug to assess its pharmacological activity in an in-vivo study. Creams containing omoconazole nitrate were prepared on a pilot basis. The therapeutic effect of the omoconazole nitrate creams was examined in an in-vivo pharmacological dermatophytosis infection model in guinea-pigs. Creams containing 0.25% or higher concentrations of omoconazole nitrate resulted in significant inhibition compared with no treatment and with vehicle-treated controls. In the mycological examination no growth of dermatophytes was observed for creams containing 1% or higher concentrations. In an in-vitro hairless mouse skin-permeability test a non-linear least squares program based on a fast inverse Laplace transform algorithm was used to calculate the partition and diffusion parameters of omoconazole nitrate in the stratum corneum and viable epidermis. The time-course of drug concentrations in the skin of the guinea-pig, estimated on the basis of these parameters, led to predictions that percutaneous drug concentrations on the guinea-pig would require 10 or more days to reach equilibrium in the skin; that drug concentrations in the corneum-viable epidermis border, where dermatophytes are considered to grow, would exceed the minimum effective concentration when 0.1% higher concentration creams were used; and that for binding to keratin drug concentrations would reach the practical minimum effective concentration when creams containing 0.5% or more omoconazole nitrate were used. These results show that partition and diffusion parameters obtained from in-vitro skin permeation studies can be used to predict in-vivo percutaneous pharmacokinetics and to estimate therapeutically effective concentrations.     

Measurement and analysis of unbound drug concentrations

The plasma protein binding of drugs has been shown to have significant effects on numerous aspects of clinical pharmacokinetics and pharmacodynamics. In many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the unbound fraction of drug in plasma which is available for distribution, elimination, and pharmacodynamic action. Thus, accurate determination of unbound plasma drug concentrations is essential in the therapeutic monitoring of drugs. Many methodologies are available for determining the extent of plasma protein binding of drugs, however, in the clinical evaluation of drug therapy, equilibrium dialysis and ultrafiltration are the most routinely utilised methods. Both of these methods have been proven to be experimentally sound and to yield adequate protein binding data. Furthermore, the characterisation of the interactions between drug and protein molecules is essential for the assessment of the pharmacokinetic implications of drug-protein binding. Protein binding parameters which characterise the affinity of the drug-protein association, the number of classes of binding sites, the number of binding sites per class or protein and the binding capacity are useful for predicting unbound drug concentrations. Simple graphical methods have often been used to obtain protein binding parameters, but these methods have limitations and are not useful for drugs with more than 1 class of binding site. Therefore, the fitting of protein binding models which characterise the drug-protein binding interaction for experimental data is the preferred method of calculating binding parameters. Using the appropriate model, values for binding parameters are typically estimated by using nonlinear least-squares regression analysis.     

Simple Metal Sorption Model for Heterogeneous Sorbents: Application to Humic Materials

A semiempirical equilibrium model to simulate proton and metal binding to heterogeneous sorbents is presented. In the simple metal sorption (SiMS) model, proton and metal binding reactions to a heterogeneous surface are conceptualized as reactions with a single, composite “site,” with empirical correction factors to the equilibrium constants that are represented as simple power functions of hydrogen ion concentration, metal-to-ligand ratio (MeT∕LT), and ionic strength (I). That is, the observed metal-binding equilibrium constant, KMe,app, is represented as KMe,app = KMe{H+}α(MeT/LT)βI?. The validity of this approach is tested by fitting the model to a hypothetical multiligand data set and three data sets from the literature involving proton and metal binding to humic materials (two data sets involving Cu2+ and H+ binding, and one data set for binding of Co2+ and H+). Independent data sets involving Cu2+ binding are used for model prediction. The fitted models are used to contrast the three humic materials in terms of acid∕base characteristics and H+∕Me exchange ratios. A theoretical limitation of the model is that it does not satisfy the Gibbs-Duhem equation for thermodynamic consistency. The major advantages of the SiMS model are simplicity (i.e., few fitting parameters), flexibility in describing proton and metal binding to heterogeneous sorbents, and ease of application (model results presented in this paper were done on a standard spreadsheet). The model is presented not as a new development in the conceptual understanding of metal-humate interactions, but rather a practical engineering tool that can easily be incorporated into general fate and transport models.     

Preliminary results of a phase II study of high-dose radiation therapy and neoadjuvant plus concomitant 5-fluorouracil with CDDP chemotherapy for patients with anal canal cancer: a French cooperative study

The mechanism of modulation of [3H]raclopride binding to dopaminergic receptors in rat brain striatal membranes by sodium ions was studied by means of equilibrium and kinetic measurements. Among different mono- and divalent cations studied, only sodium and lithium ions significantly enhanced [3H]raclopride binding to rat striatal membranes, but the effect of lithium was considerably smaller if compared with that of sodium. The equilibrium binding studies revealed that the increase in Na+ concentration from 0.5 to 150 mM increased both the radioligand affinity and the number of binding sites. The meaning of these changes was established by kinetic studies, which yielded hyperbolic plots of [3H]raclopride binding rate constants over the radioligand concentration. These plots correspond to the two-step ligand binding reaction mechanism, involving fast binding equilibrium followed by a slow isomerization of the receptor-antagonist complex. Sodium ions did not influence the antagonist affinity for the receptor sites in the first step of the binding process, nor the rate of isomerization of the receptor-ligand complex, but slowed down the rate of deisomerization. This led to a change in the value of the receptor-ligand dissociation constant Kd determined under equilibrium conditions. The same change in deisomerization rate was also sufficient to alter the receptor density (Bmax), measured by the conventional ligand binding procedure.     

Computer calculation of multiple binding equilibrium isotherms: application to the binding of bivalent ligands to antibodies interacting with cell surface Fc-receptors

A method to calculate multiple binding equilibria by looking for a set of complexes satisfying the conservation principle among sets of concentrations of ligands, receptors satisfying the mass action equations is described. The method replaces complex analytical derivations of equations representing the interactions by the minimization of a single function. The method was implemented for use on microcomputers and applied to the calculation of the binding isotherms of the interactions between a bivalent ligand, a bivalent antibody and the cell surface Fc-receptor. The binding parameters were adjusted to experimental data obtained with P388D1 cells, a monoclonal antibody against DTPA-indium complexes and monovalent and bivalent DTPA-indium haptens. The binding of the antibody and of the haptens to P388D1 cells, as a function of antibody or hapten concentration, was satisfactorily represented using a model in which the antibody molecules bind co-operatively to the Fc-receptor in the presence of cross-linking bivalent hapten. The method can thus be used as a general tool for the numeric calculation of complex equilibrium involving simultaneous interactions of multiple receptors and ligands.     

DNA sequence-specific recognition by the Saccharomyces cerevisiae "TATA" binding protein: promoter-dependent differences in the thermodynamics and kinetics of binding

The equilibrium binding and association kinetics of the Saccharomyces cerevisiae TATA Binding Protein (TBP) to the E4 and Major Late promoters of adenovirus (TATATATA and TATAAAAG, respectively), have been directly compared by quantitative DNase I titration and quench-flow "footprinting". The equilibrium binding of TBP to both promoters is described by the equilibrium TBP + DNA"TATA" left and right arrow TBP-DNA"TATA". The salt dependence of TBP binding to both promoters is identical within experimental error while the temperature dependence differs significantly. The observed rate of association follows simple second-order kinetics over the TBP concentration ranges investigated. The salt and temperature dependencies of the second-order association rate constants for TBP binding the two promoters reflect the dependencies determined by equilibrium binding. The TBP-E4 promoter interaction is entropically driven at low temperature and enthalpically driven at high temperature while the TBP-Major Late promoter reaction is entropically driven over virtually the entire temperature range investigated. These data suggest that the reaction mechanisms of TBP-promoter interactions are TATA sequence-specific and provide for differential regulation of promoters as a function of environmental variables.     

Assessment of dynamic neurotransmitter changes with bolus or infusion delivery of neuroreceptor ligands

To describe the effect of endogenous dopamine on [11C]raclopride binding, we previously extended the conventional receptor ligand model to include dynamic changes in neurotransmitter concentration. Here, we apply the extended model in simulations of neurotransmitter competition studies using either bolus or bolus-plus-infusion (B/I) tracer delivery. The purpose of this study was (1) to develop an interpretation of the measured change in tracer binding in terms of underlying neurotransmitter changes, and (2) to determine tracer characteristics that maximize sensitivity to neurotransmitter release. A wide range of kinetic parameters was tested based on existing reversible positron emission tomography tracers. In simulations of bolus studies, the percent reduction in distribution volume (deltaV) caused by a neurotransmitter pulse was calculated. For B/I simulations, equilibrium was assumed, and the maximum percent reduction in tissue concentration (deltaC) after neurotransmitter release was calculated. Both deltaV and deltaC were strongly correlated with the integral of the neurotransmitter pulse. The values of deltaV and deltaC were highly dependent on the kinetic properties of the tracer in tissue, and deltaV could be characterized in terms of the tissue free tracer concentration. The value of deltaV was typically maximized for binding potentials of approximately 3 to 10, with deltaC being maximized at binding potentials of approximately 1 to 2. Both measures increased with faster tissue-to-blood clearance of tracer and lower nonspecific binding. These simulations provide a guideline for interpreting the results of neurotransmitter release studies and for selecting radiotracers and experimental design.     

Thermodynamics of the interaction of the Escherichia coli regulatory protein TyrR with DNA studied by fluorescence spectroscopy

Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site. The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR. Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding. Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS. The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1. Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature. The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1. Several conclusions were drawn from these data. First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS. This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions. Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1). Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.     

Determination of the seven apparent equilibrium constants for the binding of oxygen by hemoglobin from measured fractional saturations

Subunit dissociation has to be taken into account in the determination of the oxygen binding constants of hemoglobin, as described by Ackers and Halvorson in 1974. The seven apparent equilibrium constants for a particular set of conditions can be determined by using extrapolations to determine the fractional saturations YT of tetramer and YD of dimer from measured values of the fractional saturation Y of partially dissociated hemoglobin. Analytical methods are used to show that YT as a function of [O2] for tetramers can be calculated from Y of hemoglobin by linear extrapolation of measured Y values at high [heme] versus [heme]-1/2 to [heme]-1/2 = 0. YD for dimers can be calculated from measured Y values by linear extrapolation of Y versus [heme] to [heme] = 0 if sufficiently low [heme] can be used. These extrapolations have been tested with numerical calculations of Y for a particular hemoglobin as a function of [heme] and [O2] by using the seven apparent equilibrium constants determined by Mills, Johnson, and Ackers in 1976. The proposed procedure also yields the apparent association constant K" for 2TotD = TotT, where TotD is the sum of the dimers and TotT is the sum of the tetramers. This thermodynamic analysis of experimental data to determine the seven apparent equilibrium constants is independent of the model used to interpret the values of the thermodynamic parameters.     

Binding of codeine, morphine, and methadone to human serum proteins

The binding properties of codeine, morphine (as representative opium alkaloids), and methadone (a synthetic pharmacologically similar compound) were studied with selected human serum proteins. The methodology involved equilibrium and dynamic dialysis using 3H-and/or 14C-labeled compounds. For estimation of the percent binding with equilibrium dialysis, concentrations of the ligand used were approximately therapeutic blood levels and another concentration 30-60 times higher. The percent binding to whole human serum ranged from about 20% for morphine to almost 60% for methadone. Of the human serum proteins investigated, the highest percent binding was found with albumin, except for methadone for which it was beta-globulin III. The affinity for other serum proteins varied with the ligand. In studies with albumin using dynamic dialysis, the plots of nubar divided by free concentration versus nubar were similar for all three ligands studied and had positive slopes, unlike those reported for acidic compounds for which the slope is always negative. In studies of binding of one ligand in the presence of another, significant competition was demonstrated, suggesting that the same binding sites were involved.     

Studies on the interaction of diflunisal ion with cyclodextrins using ion-selective electrode potentiometry

The interaction of diflunisal ion (DF) with beta-cyclodextrin (betaCD), gamma-cyclodextrin (gammaCD), and hydroxypropyl-beta-cyclodextrin (HPbetaCD) was studied in phosphate buffer, pH 7.4, at 5-37 degrees C and various CD concentrations using a home-made diflunisal ion-selective electrode. Typical direct binding plots and Scatchard plots were obtained with HPbetaCD. The Scatchard model for one class of binding sites was used for the estimation of binding parameters for the DF/HPbetaCD interaction. The estimates for n (number of binding sites per CD molecule) were in all cases very close to unity, indicating 1:1 complexation. The association constant (K) estimates decrease with increasing temperature. Sigmoidal direct binding plots and concave-downwards Scatchard plots were obtained with various betaCD or gammaCD concentrations. The Hill model was used for the estimation of the binding parameters for the DF/betaCD and DF/gammaCD interactions. Both the Hill coefficients and the binding constants were markedly dependent on the CD concentration. These findings indicate the cooperative character of DF/betaCD and DF/gammaCD interactions. The free energy change, DeltaG, and the thermodynamic parameters, DeltaH and DeltaS, were estimated for each of the interactions studied using the Van't Hoff equation.     

The affinity of human serum albumin for [3H]-digitoxin is dependent on albumin concentration

Binding of [3H]-digitoxin to human serum albumin and human serum was investigated in order to characterize the relationship between binding and albumin concentration. Binding was determined by equilibrium dialysis at 37 degrees, 24 hr was required to reach equilibrium. Volume shift and protein dilution were avoided by adding dextran 70 to the buffer compartment. [3H]-Digitoxin binding both to purified albumin and to normal serum was markedly pH-dependent, the bound/unbound ratio being highly significantly (P < 0.001) inversely correlated to pH in the range 6-8.5. When albumin concentration was increased within the physiological range, the ratio bound/unbound [3H]-digitoxin increased much less than expected from predictions using the law of mass action. Binding saturation experiments revealed that the equilibrium dissociation constant for [3H]-digitoxin was increased at higher albumin concentrations without any decrease in the number of binding sites per albumin molecule. In conclusion, the results strongly indicate that binding estimates in therapeutic monitoring of digitoxin in patients with elevated or reduced albumin concentration should not be based on the law of mass action but on empiric relationships between albumin concentration and binding.     

VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo

Three approximate free energy calculation methods are examined and applied to an example ligand design problem. The first of the methods uses a single simulation to estimate the relative binding free energies for related ligands that are not simulated. The second method is similar, except that it uses only first derivatives of free energy with respect to atomic parameters (most often charge, van der Waals equilibrium distance, and van der Waals well depth) to calculate free energy differences. The last method PROFEC (Pictorial Representation of Free Energy Components), generates contour maps that show how binding free energy changes when additional particles are added near the ligand. These three methods are applied to a benzamidine/trypsin complex. They each reproduce the general trends in the binding free energies, indicating that they might be useful for suggesting how ligands could be modified to improve binding and, consequently, useful in structure-based drug design.     

An extended pharmacokinetic/pharmacodynamic model describing quantitatively the influence of plasma protein binding, tissue binding, and receptor binding on the potency and time course of action of drugs

An extended pharmacokinetic/pharmacodynamic (PK/PD) model is presented, in which the effect of binding of the drug to plasma proteins and to tissue binding sites in a peripheral compartment, and nonspecific and receptor binding in the effect compartment are taken into account. It represents an extension of the classical Sheiner model, and the model proposed by Donati and Meistelman. The present model is characterized by the following parameters: Kue (exit rate constant of unbound drug from the effect compartment), Pue (ratio of the unbound clearances to and from the effect compartment), fue (fraction of drug in effect compartment that is not bound to nonspecific binding sites), Kd (equilibrium dissociation constant of drug-receptor binding), and Rtot (concentration of receptor binding sites in effect compartment). The rate of association and dissociation of the drug-receptor complex can be incorporated in the model. The influence of the pharmacokinetic parameters (V1, V2, fu, fu2, CLu10, CLu20, CLu12, CLu21) and the PK/PD model parameters (kue, Pue, fue, Kd, Rtot) on various dynamic parameters is analyzed. These include potency (single dose needed to produce 90% effect, ED90), constant infusion dosing rate needed to maintain a constant effect of 90%, time to maximum effect (onset time), and duration to 90% recovery. The neuromuscular blocking agent vecuronium is used as an example. It is shown that both potency and time course of action are strongly dependent on the ratio V1/fu, CLu10, kue, Pue (at equipotent doses the time course is not affected by Pue), fue, Kd, and Rtot (only if Rtot is high), whereas they are less affected by the ratio V2/fu2, CLu20, CLu12, and CLu21. In general, the model parameters affect the ED90 and the time course of action in the same direction, e.g., an increase of V1 results in an increase of ED90 and an increase of onset time and duration. However, the unbound clearance CLu10, the intercompartmental unbound clearance CLu12 and the receptor affinity Kd have an opposite effect on ED90 and the time course parameters, e.g., an increase of CLu10 results in an increase of ED90 and a decrease of onset time and duration. This effect may be responsible for the inverse relationship between onset time and potency of neuromuscular blocking drugs observed in animal experiments and clinical studies. We demonstrate that PK/PD analysis using the traditional effect compartment model (Sheiner model) results in an apparent value of keo, which is a function of kue, fue, Kd, Rtot, as well as the unbound drug concentration in the effect compartment Cue. On the other hand, the model proposed by Donati and Meistelman gives correct values of keo (equal to the product fue.kue), but the receptor affinity Kd and the receptor density Rtot obtained by this method are apparent values, which depend on fu, fue, and Pue.     

Peptide growth factors: clinical and therapeutic strategies

Total internal reflection fluorescence microscopy has been used to investigate the binding of the soluble extracellular domain of mouse Fc gamma RII (sFc gamma RII) to an anti-trinitrophenyl monoclonal mouse IgG2b (GK14.1) specifically bound to substrate-supported planar membranes composed of dipalmitoylphosphatidylcholine (DPPC) and trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-cap-DPPE). The equilibrium dissociation constants for sFc gamma RII at GK14.1-coated TNP-cap-DPPE/DPPC planar membranes containing 0.5-25 mol% TNP-cap-DPPE were approximately 1 microM. Total internal reflection with fluorescence photobleaching recovery was used to examine the dissociation kinetics. The fluorescence recovery curves were better described as a sum of two exponentials rather than by one exponential; the rates and fractional recoveries were approximately 1 s-1 (65%) and approximately 0.1 s-1 (35%). The similarity between the values of these equilibrium and kinetic parameters to those previously measured for the binding of IgG in solution to intact mouse Fc gamma RII reconstituted into planar membranes suggests that conformational changes which may occur when IgG is constrained to a membrane surface do not significantly affect the equilibrium or kinetics of IgG-mouse Fc gamma RII binding. The stoichiometry of sFc gamma RII-GK14.1 binding was 1:4, indicating that a significant fraction of the membrane-bound antibodies were not accessible for receptor binding. Possible mechanisms that might underlay the observed heterogeneity in sFc gamma RII-IgG binding kinetics are discussed.     

Cooperative study on the radioimmunological thyrotropin determination (hTSH) in serum (author''s transl)

The increased incidence of drowsiness in hypoalbuminemic patients administered diazepam and more rapid clearance of tolbutamide in cirrhotics may be due to changes in plasma protein binding. The binding of diazepam and tolbutamide was studied by equilibrium dialysis at 37degreesC over a total drug concentration range of 1 to 10 mug/ml and 50 to 300 mug/ml, respectively, in plasma from 21 normal and 14 alcoholic subjects. At 1 mug/ml, diazepam plasma protein binding (+/- S.D.) was 98.5+/-0.4 per cent in normals and 97.8+/-1.2 per cent in alcoholics; at 100 mug/ml, tolbutamide binding was 97.8+/-0.3 per cent in normals and 95.1+/-4.2 per cent in alcoholics. For both agents at all concentrations, the binding to plasma from alcoholics was significantly decreased (P less than 0.01-less than 0.02). The extent of binding of both drugs was dependent on the albumin concentration. These findings suggest that important changes in pharmacologic effect, distribution, and clearance of diazepam and tolbutamide can be anticipated in alcoholics with hypoalbuminemia.     

Crab clocks rewound

The effect of amphetamine (AMPH), codeine (COD), methamphetamine (MEPH), morphine (MORP), and benzoylecgonine (BE) on the binding of cocaethylene (CE) and cocaine (COC) to human serum in vitro was investigated by equilibrium dialysis at 4 degrees C. Each compound was added individually at concentrations of 500, 1,000, or 2,000 nM to pooled human serum containing COC or CE at 500 nM concentration. For COC, the addition of COD, MEPH, and CE enhanced serum binding whereas MORP and BE decreased it. Variable effects on COC binding were noted for AMPH. For CE, the addition of COD and COC generally increased binding whereas MORP decreased it. No appreciable effect on CE binding was observed after adding AMPH, MEPH, and BE. Except for CE, AMPH, and MEPH in the presence of COC, the binding of COC and CE tended to be less with 2,000 nM of each added drug than at lower concentrations of them, presumably because of mass-action displacement of COC and CE at the higher concentration. These findings should be clinically important because these drugs are frequently found together in patients.     

Late outcome of amputees with premature atherosclerosis

Plasma free T4 (FT4) concentrations could be increased during hemodialysis in patients with chronic renal failure (CRF) because an increase in non-esterified fatty acids (NEFA) could interfere with the binding of T4 to thyroxine-binding globulin. To evaluate the effect of hemodialysis on the FT4 concentration in patients with CRF, we measured the FT4 in 39 patients with CRF by four assay methods including equilibrium dialysis, the 125I-T4 analog method and enzyme immunoassay. The addition of the fatty acid sodium oleate to normal pooled sera led to a marked increase in FT4 as measured by equilibrium dialysis (Model FT4). A moderate increase in the serum FT4 concentration also was observed with an IMX enzyme immunoassay kit, whereas the Coat-A-Count analog method demonstrated no interference by sodium oleate. The mean serum FT4 prior to hemodialysis measured by equilibrium dialysis did not differ significantly from that in the normal control, although those measured by analog methods (Coat-A-Count and Amerlex) and IMX were subnormal. The FT4 by IMX were albumin-dependent, and the values decreased as the samples were serially diluted, but Model FT4 was not affected by the albumin level or the serial dilution. FT4 by Model FT4 showed a marked increase beginning 10 min after the start of dialysis, and it correlated well with the plasma concentration of NEFA and the NEFA/albumin molar ratio. The other three assay methods, including one which is not affected by NEFA, did not show a change in FT4 at 10 min, but a significant increase of 11 to 17% was observed by the end of dialysis. The TSH concentration decreased significantly during hemodialysis. These data suggest that (1) the low serum FT4 in hemodialysis patients measured by some immunoassay methods may be an underestimation due to the low albumin level; (2) FT4 actually increases during hemodialysis due to the actual increase in NEFA, although the marked increase in FT4 during hemodialysis as measured by equilibrium dialysis is an overestimation due to the in vitro generation of NEFA; and (3) one should beware of aberrations in thyroid hormone parameters during hemodialysis and potential complications.