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Biomedical subjects

R Brodersen

Publications and source records attributed to R Brodersen.

At least 19 recordsLinked to original sources
PubMed1992

Solubility of long-chain fatty acids in phosphate buffer at pH 7.4.

The solubility of the saturated fatty acids lauric, myristic, palmitic, and stearic acid and the unsaturated oleic acid at 37 degrees C in phosphate buffer (pH 7.4) was estimated by using two independent methods. The one was a conventional solubility technique measuring the concentration of dissolved fatty acid in buffer by using radioactive compounds. The other was a dialysis exchange technique monitoring possible aggregation of solvated fatty acid anions by measuring the rate of diffusion of labelled compound across a dialysis membrane under conditions of chemical equilibrium. It was found that the results were strongly dependent on the radiochemical purity of the fatty acids. Using highly purified samples of radioactively labelled fatty acids, the solubility of monomeric laurate was shown to be greater than 500 microM, whereas the solubility of monomeric myristate was found to be 20-30 microM. Palmitate, stearate, and oleate solutions, on the other hand, showed a tendency to aggregation even at concentrations below 1 microM. Special attention was given to palmitate, as a reference compound for long-chain fatty acids, and the solubility of monomeric palmitate was estimated to be lower than 10(-10) M.

Autoradiography↗
PubMed1992

Ionization of tyrosine residues in human serum albumin and in its complexes with bilirubin and laurate.

Spectrophotometric titration of human serum albumin indicates that ionization of the 18 tyrosine residues takes place between pH 9 and 12.7. A Hill plot indicates that protons dissociate co-operatively from tyrosine residues, in pure albumin between pH 11.0 and 11.4 with a Hill coefficient 1.7, and in the bilirubin-albumin complex between pH 11.2 and 11.7 with a Hill coefficient 1.6. With a stopped-flow technique it is shown that about seven of the tyrosines ionize fast, with rate constants well above 10(2) s-1, when pH is suddenly changed from near neutral to pH 11.76. Further residues ionize slowly, with rate constants around 10(2) s-1 or less. The N-form of albumin (pH 6) contains one more fast ionizing tyrosine than the B-form of albumin (pH 10). Binding of bilirubin or laurate to the albumin molecule (molar ratio 1:1) transforms one to three of the fast ionizing tyrosines to slowly ionizing.

Bilirubin↗
PubMed1992

The effect of chloral hydrate and its metabolites, trichloroethanol and trichloroacetic acid, on bilirubin-albumin binding.

Chloral hydrate is used as a sedative in infants requiring ventilatory support. The metabolites, trichloroethanol and trichloroacetic acid, accumulate in the serum and are protein bound. The possibility that these chemicals might compete with bilirubin for albumin binding was tested using the peroxidase method and a dialysis rate method. Chloral hydrate and trichloroethanol had no effect on bilirubin-albumin binding. Trichloroacetic acid affects bilirubin-albumin binding but to a degree that would be dangerous only in infants with an unusual accumulation of this metabolite.

Bilirubin↗
PubMed1991

Binding of long-chain fatty acids to serum albumin in healthy humans. Relationship to obesity.

Equilibria of the binding of palmitate to serum albumin in adults are studied by dialysis-exchange-rate determinations. The results are used for a description of binding equilibria of fatty acids in general, as follows. 1. The reserve albumin concentration, p, for binding of palmitate is used as an approximate measure of p*, the reserve albumin concentration for binding of mixed fatty acids present in serum. 2. The total availability of fatty acids is defined as C*/p*, where C* is the total concentration of non-esterified fatty acid. 3. The fatty-acid-binding property of albumin is described by L* = p*/P = alpha C*/P, where P is the albumin concentration. The numerical value of alpha is -0.05. The above parameters are measured in sera from four healthy volunteers, in whom large variations of serum fatty acid concentration occurred. A group of 64 healthy students showed considerable variation of L* from one individual to another. It is found that L* decrease significantly with increasing body mass index (body mass divided by the square of the body length). In 42 patients with diabetes type I, L* was independent of body mass index. These findings are consistent with a previously formulated hypothesis of mechanism of obesity.

Adult↗
PubMed1991

Effect of drug combinations on bilirubin-albumin binding.

Drugs which compete with bilirubin for albumin binding may increase the risk of kernicterus. Fortunately, few drugs are strong competitors. However, in neonatology, many drugs are used simultaneously. We have studied the effect of drug combinations on bilirubin binding using human serum albumin and the peroxidase method. Combinations of aminophylline with phenobarbital, cefotaxime and vancomycin were studied as well as the combination of vancomycin and cefotaxime. The results show that the bilirubin-displacing effect of the drug combinations cannot be predicted from each drug's individual effect. These results are consistent with a flexible model of albumin binding. Combinations of drugs which are both albumin-bound and reach high serum concentrations should be tested for their combined effect on bilirubin binding and this information used in deciding on treatment in sick, premature infants.

Aminophylline↗
PubMed1990

Thermodynamic parameters for binding of fatty acids to human serum albumin.

Binding of laurate and myristate anions to human serum albumin has been studied over a range of temperatures, 5-37 degrees C, at pH 7.4. The binding curves indicate that the strength of binding of the first few molecules of fatty acid to albumin (r less than 5) decreases with increasing temperature, whereas binding of the following molecules seems to proceed independently of temperature. Binding data were analyzed according to the general binding equation yielding several sets of acceptable binding constants within a probability limit of 0.75. From the temperature dependence of the first step constant, it was possible to calculate values for the changes in enthalpy and entropy during the initial binding step. For the medium-chain fatty acids, laurate and myristate, binding of the first molecule to albumin appeared to be enthalpic, with a tendency to an increasing contribution of entropy to binding energy with increasing chain length of the fatty acid.

Humans↗
PubMed1990

Multiple fatty acid binding to albumin in human blood plasma.

Binding equilibria of long-chain fatty acids to human serum albumin, in serum or plasma, were studied by a dialysis exchange rate technique. Palmitate was added to citrated plasma in vitro and it was observed that between six and ten palmitate molecules were bound to albumin with nearly equal affinity. Observations in vivo gave similar results in the following series: (a) in two volunteers with increased fatty acid concentrations after fasting, exercise, and a cold shower: (b) in three male volunteers in whom high concentrations of non-esterified fatty acids, up to 4.6 mM, were induced by intravenous administration of a preparation of lecithin/glycocholate mixed micelles, and (c) in 81 patients with diabetes mellitus, type I. The binding pattern of palmitate in serum or plasma is essentially different from that observed with palmitate added to buffered solutions of pure albumin when two molecules are tightly bound and about four additional molecules with lower affinity. The differences may partly be explained by the presence of chloride ions in blood plasma, reducing the affinity for binding of the first two fatty acid molecules, and partly by facilitated binding of several molecules of mixed fatty acids, as found in plasma.

Diabetes Mellitus, Type 1↗
PubMed1990

Two-dimensional electrophoresis of the fatty acid binding protein from human heart: evidence for a thiol group which can form an intermolecular disulfide bond.

A 100,000 g supernatant from human heart muscle, containing cytosolic proteins with some contaminating plasma proteins, was analyzed for fatty acid binding protein (FABP) by two-dimensional electrophoresis (2-DE) using isoelectric focusing under nondenaturing conditions in the first dimension. FABP purified from human heart muscle was found to comigrate with a major spot in 2-DE gels of the supernatant. This spot was comparable with those of the myoglobins in staining intensity. When purified FABP was charged with [3H]palmitate and subjected to nondenaturing 2-DE, radioactivity always comigrated with this protein. Under denaturing and reducing conditions in the second dimension, FABP was found to have a pI of 5.3 and an apparent molecular weight of 15,000. Isoforms of FABP, reported here for the first time to occur in human heart muscle, were observed as minor spots focusing at pH 5.1 and 5.7. When electrophoresis in the second dimension was carried out under denaturing but nonreducing conditions, an additional protein appeared at pH 5.3 with an apparent molecular weight of about 30,000. This protein was identified as a dimer of FABP and evidence for the involvement of an intermolecular disulfide bond in this dimerization is presented.

Carrier Proteins↗
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

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

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↗
PubMed1979

Bilirubin. Solubility and interaction with albumin and phospholipid.

Bilirubin is generally considered a lipophilic substance, and its neurotoxicity is ascribed to an affinity for lipids in the central nervous system. In the present paper, it is shown that the solubility of bilirubin in apolar solvents and in triglycerides is low and increases with solvent polarity. Consequently, bilirubin should not be characterized as lipophilic. The solubility in aqueous buffers was studied under exclusion of light and was found lower than previously reported, about 7 nM at pH 7.4, temperature 37 degrees C, increasing with higher pH, approximately in inverse proportion with the squared hydrogen ion concentration. Binding of bilirubin to human serum albumin was studied by the rate of oxidation with peroxidase of the free ligand at equilibrium. The stoichiometry of proton involvement in the binding process was investigated by acidimetric titration. It is concluded that precipitation of bilirubin in vivo is thermodynamically possible. It was further demonstrated by light absorption spectroscopy that bilirubin forms a complex with phosphatidylcholine in diethyl ether and that an aqueous suspension of phosphatidylcholine enhanced aggregation of bilirubin. Transfer of bilirubin dianion from its complex with plasma albumin and precipitation of bilirubin acid with phosphatidylcholine in membranes of nerve cells is therefore possible and may account for the neurotoxicity.

Bilirubin↗
PubMed1979

Kinetics of bilirubin oxidation with peroxidase, as applied to studies of bilirubin-albumin binding.

In the determination of unbound bilirubin by rate of oxidation with peroxidase, errors may be caused by (1) phenol, propylparaben, and phenothiazines (free radical acceleration), (2) haemoglobin (peroxidase effect), and (3) ascorbate (inhibition). Such errors may be diminished by dilution 1:40, or with an anti-oxidant, tert-butyl-p-hydroxyanisole, and ascorbate oxidase.

Ascorbate Oxidase↗
PubMed1979

Bilirubin acidity. Titrimetric and 13C NMR studies.

Acidimetric titration of bilirubin IX-alpha, dissolved in excess aqueous sodium hydroxide, showed that two protons are dissociated with pK values well below 7 and that one or several additional acidic groups titrate with pK around 12.9. Precipitation of the nearly insoluble acid precluded determination of the two lower pK values by titration in aqueous solution. In dimethyl sulfoxide solution, four acidic protons were demonstrated, titrating two by two without precipitation. 13C NMR spectra of bilirubin IX-alpha were recorded and complete assignments were made by comparison with the spectra of bilirubin XIII-alpha and mesobilirubin etc. Such spectra, recorded after addition of 2 and 4 mol of base per mol of bilirubin IX-alpha, showed that both carboxyl groups are titrated by the first 2 mol of base, and both lactams by the following 2 mol of base. Cotitrations of bilirubin IX-alpha with other acids, o- and m-hydroxybenzoic acid and 2-pyridone, were used to determine relative pK values in dimethyl sulfoxide solution, and pK values for the four acidic protons of bilirubin IX-alpha in aqueous solution were calculated from the Born equation. Both carboxyl groups exhibited pK = 4.4, and both lactams pK = 13.0, in good agreement with values expected from the chemical structure of the bilirubin molecule. The implications of these findings for understanding the mechanism of bilirubin neurotoxicity are discussed.

Bilirubin↗