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Biomedical subjects

R Brodersen

Publications and source records attributed to R Brodersen.

At least 37 records · Page 2Linked to original sources
PubMed1990

Deposition of bilirubin acid in the central nervous system--a hypothesis for the development of kernicterus.

On the basis of the concentration of unconjugated bilirubin and available albumin for the binding of bilirubin it is possible to calculate the level of unbound bilirubin in a serum sample. The solubility of bilirubin can further be calculated when the pH is known. In cases of threatened kernicterus the free bilirubin concentration in serum samples from newborn infants surpasses the solubility by a factor close to one hundred. It is hypothesized that deposition of bilirubin in tissues takes place as an ongoing event, the deposited pigment being eliminated by bilirubin oxidase in healthy infants. Kernicterus results when the rate of deposition becomes overwhelming as a result of high bilirubin concentration, low albumin reserve, low pH, after administration of a displacing drug, or if the bilirubin oxidase system has been compromised by preceding birth asphyxia or other forms of central nervous system injury.

Adolescent↗
PubMed1990

Comparison of the binding characteristics of serum albumins from various animal species.

In order to develop an animal model to study the bilirubin displacing effect of various drugs, we compared the bilirubin binding ability of human, pig, dog, rabbit, hamster, rat, guinea pig, and cat albumin. These albumins were used also to study the binding of monoacetyldiaminodiphenyl sulfone (MADDS). Using human, rat, guinea pig, and rabbit albumin, we studied the effect of sulfisoxazole, ceftriaxone, and tin protoporphyrin on bilirubin binding. Our results demonstrate that each animal albumin has different binding characteristics for the various chemicals tested. This variable must be considered before using an animal as a model for studying factors influencing bilirubin deposition in the brain.

Animals↗
PubMed1990

Valproate and palmitate binding to human serum albumin: an hypothesis on obesity.

Binding equilibria of valproate (2-n-propyl-pentanoic acid anion) with defatted human serum albumin were studied by equilibrium dialysis in a 66 mM sodium phosphate buffer, pH 7.4, 37 degrees. Three hundred and fifty-six observed points for bound versus free valproate concentration were obtained and analyzed in terms of stepwise binding. It was found that the best fit resulted from a model in which 67% of the albumin was capable of binding valproate, whereas 33% did not bind. Thirty acceptable variants of the curve fitting were generated in order to assess the variation of the binding constants. The binding albumin component combines with three molecules of valproate with high affinity and with at least seven additional molecules that are loosely bound. Saturation of the protein cannot be reached. At very high concentrations of free valproate (above 10 mM) irreversible changes in the albumin take place, resulting in poor reproducibility in the amount of bound valproate. In the presence of palmitate, 0.5, 1, and 1.5 mol/mol of albumin, binding of valproate is decreased by a competitive mechanism. It is hypothesized that obesity, developing as a complication of valproate treatment of epilepsy, results from increased availability of long-chain fatty acids due to competitive valproate binding.

Humans↗
PubMed1990

Increase of plasma nonesterified fatty acid concentration and decrease of albumin binding affinity after intravenous injection of glycocholate-lecithin mixed micelles.

Lipophilic drugs intended for intravenous use can be solubilized by mixed-micellar systems containing glycoholic acid and lecithin (MM). Our present studies determined the influence of such MM preparations on albumin binding of monoacetyldiaminodiphenyl sulfone (MADDS), a deputy ligand for bilirubin. After intravenous administration of MMs to healthy male and female adult volunteers, concentration-time profiles of bile acid and nonesterified (NEFA) and esterified fatty acids were obtained as well. In vitro experiments with blood from adults and from neonatal cords indicated a modest reduction in reserve albumin for binding of MADDS after addition of MMs, resulting from glycocholic acid in the micellar preparation. After injection of MM preparations with up to 530 mg glycocholic acid, a rapid decrease of the reserve albumin was observed. The effect was more pronounced in men than in women and resulted in different areas under the time curve for the decrease (p = 0.049). At their maximum (3 to 10 minutes after MM doses) the decreases averaged (+/- SD) 68% +/- 14% in men and 45% +/- 8.5% in women. Low reserve albumin concentrations were maintained over 20 minutes despite rapidly declining bile acid concentrations. Injection of MM caused a drastic increase of NEFA in the serum samples with a more pronounced effect in men (average +/- SD increase: 473% +/- 93%) than in women (148% +/- 91%) (p = 0.01). The changes in NEFA concentrations ran reciprocal to the changes of reserve albumin for binding MADDS. In all subjects the increase in NEFA was accompanied by a decrease in reserve albumin for palmitate. Fatty acid binding to albumin was well restored within 1 hour. Thus, before drugs incorporated in MM can be prescribed to neonates who are at risk for having kernicterus, the impact of intravenous MM on bilirubin binding and NEFA levels must be investigated in that patient population.

Adult↗
PubMed1989

Serum albumin binding of palmitate and stearate. Multiple binding theory for insoluble ligands.

In usual studies of ligand binding to a carrier, free and bound ligand concentrations are measured in equilibrium mixtures with varying carrier and ligand concentrations. The observed data are then analyzed by a binding equation such as Scatchard's or the general binding equation. With palmitic, stearic and oleic acids as ligands we found that the aqueous solubility is too low to allow this procedure. We have consequently transformed the general binding equation so that it does not contain parameters related to aqueous solutions of the ligand. While the classical binding equations describe affinities of transfer of a ligand from an aqueous solution to the carrier, the new equation is valid for transfer of a ligand from one bound state to another, i.e. for relative binding description. The relative binding constants, L1, L2, L3 ... Li, in the new equation thus define the transfer affinity for the ligand from a 1:1 complex with a standard carrier to an i:1 complex of the ligand with the carrier investigated. Binding of palmitate and stearate to human serum albumin was studied by determination of dialytic exchange rates between identical fatty acid/albumin solutions. The results were analyzed by the new equation without reference to ligands in aqueous solution.

Chlorides↗
PubMed1989

Drug binding properties of neonatal albumin.

Neonatal and adult albumin was isolated by gel chromatography on Sephacryl S-300, from adult and umbilical cord serum, respectively. Binding of monoacetyl-diamino-diphenyl sulfone, warfarin, sulfamethizole, and diazepam was studied by means of equilibrium dialysis and the binding data were analyzed by the method of several acceptable fitted curves. It was found that the binding affinity to neonatal albumin is less than to adult albumin for monoacetyl-diamino-diphenyl sulfone and warfarin. Sulfamethizole binding to the neonatal protein is similarly reduced when more than one molecule of the drug is bound per albumin molecule, and binding of the first sulfamethizole molecule is possibly reduced as well. Diazepam binds with equal affinity to the fetal and adult proteins. Among the two main albumin drug-binding functions, for warfarin and diazepam, the former is thus compromised in the newborn infant while the diazepam binding function is at the adult level.

Adult↗
PubMed1989

Skin colour and bilirubin in neonates.

The correlation between the yellow colour of the skin and serum bilirubin concentration, reserve albumin concentration, and pH was investigated in 76 icteric neonates. Significant linear correlation existed between yellow colour of the skin and serum bilirubin concentration, reciprocal of the reserve albumin concentration, and the squared hydrogen ion concentration. Furthermore, the basic yellowness of the skin at birth correlated linearily with the yellow colour of the skin measured when the child became jaundiced. The results support the proposed hypothesis that bilirubin is transferred from plasma to skin through two different mechanisms: (a) leakage of bilirubin-albumin complexes into extravascular spaces and (b) precipitation of bilirubin acid in phospholipid membranes. The latter mechanism suggests that measurement of the yellow colour of the skin may be a better predictor of brain damage than the serum bilirubin concentration and thus be of clinical utility. Measurement of the yellow colour of the skin as a method of obtaining serum bilirubin concentration is unreliable.

Bilirubin↗
PubMed1989

Displacement of bilirubin from adult and newborn serum albumin by a drug and fatty acid.

Competitive binding of bilirubin and 5-butyl-1-cyclohexylbarbituric acid (bucolome) to human serum albumin was investigated by equilibrium dialysis, with and without added bilirubin (0.5 mol/mol albumin). Further observations were made by peroxidase oxidation kinetics. Finally, cobinding of the two ligands was studied by spectrophotometry. Experiments were performed with defatted adult human serum albumin, with umbilical cord serum, and with adult albumin to which was bound 2 mol palmitate/mol albumin. Bucolome does not induce displacement of bilirubin from binding to defatted adult albumin when one molecule of either ligand is bound. The second molecule of bound bucolome displaces the first bilirubin molecule. With albumin in umbilical cord serum, the first bucolome does decrease binding of the first bilirubin to some extent although the two ligands can bind to the same albumin molecule. The presence of two molecules of bound palmitate together with one of bilirubin causes enhanced bilirubin displacement on binding of bucolome. These observations may be of consequence for practical testing of the bilirubin displacing effect of drugs. Experiments with neonatal albumin and with added fatty acid should be included for a complete study.

Adult↗
PubMed1989

Ceftriaxone binding to human serum albumin: competition with bilirubin.

Ceftriaxone, a cephalosporin, is bound reversibly to defatted human serum albumin from adults, with a first stoichiometric binding constant of 60,000 M-1, as found by equilibrium dialysis at pH 7.4, 37 degrees. A second molecule is weakly bound, with a binding constant of 500 M-1. Possible cobinding with bilirubin was studied by the peroxidase method and by equilibrium dialysis with and without added bilirubin. Results indicated competitive binding; formation of an albumin complex containing both bilirubin and ceftriaxone could not be demonstrated. Light absorption spectra of bilirubin-albumin showed little change on addition of ceftriaxone, in agreement with the competitive biding mechanism. Binding to serum albumin from newborn infants is weaker than to the protein from adults, with the first binding constant being about 36,000 M-1. Cobinding of ceftriaxone and bilirubin to albumin from newborn infants is likewise competitive. It is concluded that ceftriaxone is a strong bilirubin displacer with a potential of inducing bilirubin encephalopathy in predisposed newborns.

Bilirubin↗
PubMed1988

Myristic acid binding to human serum albumin investigated by dialytic exchange rate.

Dialysis rate determinations of several fatty acids in the absence of albumin revealed that the myristate anion, like that of laurate, in aqueous solution, pH 7.5, is present as a monomer anion when the concentration is below 25 microM. Palmitate and oleate solutions, on the other hand, show a tendency to aggregation even at concentrations below 0.5 microM. Multiple binding of myristate to human serum albumin in phosphate buffer, at pH 7.5, 37 degrees C, was investigated by exchange of 14C-labeled myristate across a dialysis membrane under conditions of binding equilibrium. A binding isotherm was established by least squares fitting of the stoichiometric binding constants in the stepwise binding equation to the experimental data. The best-fit solution was supplemented with 30 acceptable solutions within a probability limit of 0.95. A concept of one or two distinct high-affinity sites for binding of fatty acids could not be verified; the observations allow a variety of binding mechanisms ranging from cooperativity of the first two myristates to a model with four equal and independent sites.

Dialysis↗
PubMed1988

Palmitate binding to serum albumin, measured by rate of dialysis.

Dialysis experiments were performed with an acetylcellulose membrane between two identical sample solutions; a trace amount of radiolabelled palmitate was added on one side and the rate of dialytic equilibration of the label was measured. By comparison with rates measured in standard experiments, using pure albumin solutions, we obtained the reserve albumin concentration for binding of palmitate, previously defined as the concentration of a pure standard albumin which binds the labelled ligand as tightly as it is bound in the sample. Two techniques were developed, one for 1-ml sample volumes, another for 25 microliter. Reserve albumin for binding of palmitate, measured in pure albumin solutions, decreased equally on addition of palmitate and stearate, slightly less with oleate and still less with linoleate, indicating that palmitate and stearate are bound competitively while interaction with binding of the unsaturated acids is less pronounced. Chloride ions compete with binding of palmitate. Reserve albumin concentration in serum samples from 33 male adults was 420 +/- 59 microM (mean +/- SD), and in 33 females, 351 +/- 50 microM. Umbilical cord sera from ten newborn infants averaged 172 +/- 56 microM. The reserve albumin concentrations in the sera of newborn infants are low compared with the normal albumin concentration, which is about 600 microM. This appears to indicate the presence of an albumin species with lower affinity for palmitate.

Adult↗
PubMed1988

Detection of carrier heterogeneity by rate of ligand dialysis: medium-chain fatty acid interaction with human serum albumin and competition with chloride.

Binding equilibria for decanoate, octanoate, and hexanoate to defatted human serum albumin were investigated by dialysis exchange rate determinations in 66 mM sodium phosphate buffer, pH 7.4, 37 degrees C. The binding isotherms for decanoate and octanoate could not be fitted by the general binding equation. It was necessary to assume the presence of two albumin components, one with high affinity and one with low affinity, about 0.65 of the albumin having high binding affinity. The first stoichiometric binding constants for the high- and low-affinity albumin components were 1.1 X 10(7) and 1.4 X 10(5) M-1, respectively, for decanoate; 1.6 X 10(6) and 3.5 X 10(4) for octanoate; and 7.1 X 10(4) and 8.0 X 10(2) M-1 for hexanoate. The high-affinity albumin component binds 1 mol decanoate, 1 mol octanoate, or 2 mol hexanoate more than is bound to the low-affinity component. Chloride ions compete with the high-affinity binding of all three ligands. Albumin dimer, present in the commercial human serum albumin, has approximately the same binding properties as the monomer. Mercaptalbumin, isolated from the preparation, also consists of two proteins, with first stoichiometric binding constants 8.0 X 10(6) and 1.4 X 10(5) M-1 for decanoate, approximately 0.5 of the mercaptalbumin having high affinity.

Binding, Competitive↗
PubMed1988

Multiple binding of bilirubin to human serum albumin and cobinding with laurate.

Numerical analysis of multiple binding of two ligands to one carrier has been accomplished, using the principle of several sets of acceptable binding constants, with bilirubin-laurate-albumin as an example. Binding of bilirubin to defatted human serum albumin was investigated by a spectroscopic method, based upon a difference of light absorption spectrum for free and bound bilirubin. The observations were supplemented with previous data from an independent technique, measurement of oxidation rates of free bilirubin with hydrogen peroxide and peroxidase. A continuous isotherm was obtained, showing binding of at least 4 mol bilirubin per mole albumin with the following stoichiometric binding constants, 1.11 X 10(8), 1.7 X 10(7), 8 X 10(5), and 4 X 10(4) M-1 at pH 8.2, ionic strength 0.15 M, 25 degrees C. The binding is anticooperative at all steps. A saturation level was not reached. Cobinding of bilirubin and laurate was studied, with up to 2 mol of each ligand per mole albumin, using the peroxidase method for determination of free equilibrium concentrations of bilirubin, and a dialysis rate technique for free laurate. The findings could be described in terms of a stoichiometric model. Heterotropic cooperativity was present among the first bilirubin and the first and second laurate molecules. More than two molecules of either ligand can be bound at the same time.

Bilirubin↗
PubMed1988

Cobinding of bilirubin and laurate to human serum albumin: spectroscopic characterization of stoichiometric complexes.

Light absorption and CD spectra of bound bilirubin and albumin fluorescence spectra have been recorded from mixtures containing albumin, A, bilirubin, B, and laurate, L, in Tris-NaCl buffer at pH 8.2, 25 degrees C. Concentrations of the corresponding stoichiometric complexes, ABiLj, for i = 0/3 and j = 0/3, have been calculated from previously determined stoichiometric cobinding constants (H. Sato et al. (1988) Arch. Biochem. Biophys. 260, 811-821). Spectral data of the complexes have finally been found by iterative computer fitting using the principle of several acceptable solutions (R. Brodersen et al. (1987) Eur. J. Biochem. 169, 487-495). The results were utilized at the microscopic level to investigate ligand-induced conformational changes. When laurate was bound to AB, a decrease of the distance between Trp-214 and the bound bilirubin occurred, as measured according to Förster's principle. The distances were 21.9 +/- 0.3 A in AB, 19.7 +/- 0.3 A in ABL, and 17.9 +/- 0.2 A in ABL2.

Bilirubin↗
PubMed1988

Albumin binding of MADDS--a measure of bilirubin binding--in women during pregnancy and after delivery and in their infants.

The purpose of this study was to investigate the binding potential of MADDS (monoacetyldiaminodiphenyl sulphone) to albumin, a measure for binding of unconjugated bilirubin, in healthy women during pregnancy, during and after delivery, and in their infants. The serum concentrations of unconjugated bilirubin, reserve albumin for binding of MADDS and total albumin were measured in: (a) 21 non-pregnant women; (b) 16 pregnant women in the 16th-24th, 28th-32nd, and 36th-38th gestational weeks, and at the time of delivery from both mother and infant; and (c) 15 women at the time of delivery, and 24 and 72 hours after delivery. The bilirubin concentrations did not change during pregnancy or at delivery and were very small compared with the concentrations of reserve albumin for binding of MADDS and total albumin. Therefore, the ratio of reserve albumin to total albumin was an expression of the binding potential of the albumin for MADDS in women. During pregnancy, the reserve albumin decreased equal to total albumin, so that the ratio was not significantly changed (p greater than 0.05). In contrast, at delivery the reserve albumin was significantly lower (p less than 0.01) than in the 36th-38th gestational weeks, without any significant difference in total albumin, i.e. the ratio was significantly lower than during pregnancy (p less than 0.01). During the first 3 days after delivery the reserve albumin increased significantly (p less than 0.05), also without any significant change in total albumin, so that the ratio increased significantly (p less than 0.05) and was normalized. The binding potential for MADDS to albumin in newborn infants was even lower than that of their mothers. Since the binding-potential is reduced in both mother and infant at delivery, a relation is suggested.

Adolescent↗
PubMed1987

Characterization of binding equilibrium data by a variety of fitted isotherms.

Experimental binding equilibrium data, resulting from measurement of ligand binding to macromolecular carriers, are usually described by fitting of binding constants. These constants are often highly variable, as illustrated in the present paper by two examples, binding of salicylate to human serum albumin and of oxygen to hemoglobin. In order to avoid over-interpretation of binding constants it is pointed out that the best-fit solution, obtained by least-squares fitting, may be supplemented by several, e.g. thirty, acceptable solutions. It is further shown how the 30 sets of acceptable binding constants, plotted as Klotz' affinity profiles, can serve for evaluation of cooperative effects.

Algorithms↗
PubMed1987

Cobinding of bilirubin and sulfonamide and of two bilirubin molecules to human serum albumin: a site model.

Differential light absorption spectra of the bilirubin-albumin 1:1 complex, obtained on addition of 20 different sulfonamides, differ with respect to shape and amplitude. This finding seems to indicate that the sulfonamide molecule is bound in direct touch with the bilirubin. The light absorption spectrum of bilirubin-albumin 1:1 undergoes changes on cobinding of a fatty acid anion, laurate, and on variation of pH, previously explained by a change of dihedral angle between the two chromophores of the bilirubin molecule. In bilirubin-albumin 2:1, binding of laurate and variation of pH cause little change of the spectrum. This is best explained by binding of the two bilirubin molecules in close proximity, preventing conformational changes in the complex. From measurements of fluorescence of the lone tryptophan group in albumin and quenching on binding of bilirubin, we calculated the distance of 22 A from tryptophan to the first bound bilirubin molecule, and of 18 A to the second. Mutual quenching of the bilirubin fluorescence from two bound bilirubin molecules seemed to indicate that the two are bound closely together. A model of bilirubin-albumin with a binding site capable of accommodating one bilirubin and one sulfonamide molecule, or two molecules of bilirubin, is compatible with our findings.

Bilirubin↗