In vitro amiodarone protein binding and its interaction with warfarin

The binding of amiodarone to human plasma protein and to bovine serum albumin was studied by three different methods, ultracentrifugation, equilibrium dialysis and fluorescence spectroscopy. The fraction of amiodarone bound to plasma protein amounted to 96.3%. The changes in the binding properties of 1-anilino-naphthalene-8-sulfonate for bovine serum albumin using warfarin and amiodarone as independent inhibitors were analyzed in terms of binding site specificity. The findings indicated that amiodarone and warfarin have two different binding sites on bovine serum albumin, so a noncompetitive inhibition mechanism was indicated. On the basis of our data we cannot exclude other mechanisms of interaction besides direct displacement of one drug by another; nevertheless, metabolite interference between amiodarone and coagulation cofactors may better explain the enhancement of warfarin's pharmacological action in association with amiodarone.     

On the binding of steroid sulfates to albumin

3H-Labeled steroid sulfates, sulfate of estrone (E1S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E1S). The apparent equilibrium constants (K) of the tracers did not measurably change at concentrations of the non-radioactive sulfates below 10(-5) mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of 3H-E1S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E1S in vivo, because of its preferential occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.     

Chaperone-like features of bovine serum albumin: a comparison with α-crystallin

The chaperone behaviour of bovine serum albumin was compared with that of α-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with α-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than α-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family. Received 29 August 2005; received after revision 23 September 2005; accepted 12 October 2005     

Interaction between azapropazone and warfarin

Summary In vitro studies, using 2 separate techniques, have shown that the anti-inflammatory agent azapropazone caused displacement of warfarin from its plasma albumin binding and it is therefore suggested that such a displacement mechanism may be involved in the reported clinical interaction between these 2 drugs.     

Binding of 3,3',5'-triiodo-L-thyronine (rT3) to human serum proteins]

The rT3-binding and human serum proteins was directly studied with tracer doses of radioactive rT3. Polyacrylamide gel electrophoresis showed 125I-rT3 added to human serum was distributed among two proteins: albumin (carrying 57% of tracer rT3) and TBPA )22%). No binding was observed to TBG, protein binding T4.     

Tannic acid inhibits insulin-stimulated lipogenesis in rat adipose tissue and insulin receptor function in vitro

Tannins occur naturally in relatively abundant amounts in fruits, herbal medicines and common beverages. Thus an understanding of how these polyphenols affect peptide hormone action is of importance. We report here that tannic acid (a hydrolysable tannin) inhibits insulin-stimulated lipogenesis in rat adipose tissue in vitro, with an IC₅₀ estimated to be about 350 M. However, its monomer, gallic acid, did not show a similar inhibitory effect at concentrations up to 1 mM. The inhibition by tannic acid was less evident with higher concentrations of bovine serum albumin in the incubation buffer. This was attributed to the formation of a tannin-protein complex between bovine serum albumin and tannic acid. In a binding assay, it was observed that the specific binding of insulin to its receptor was not inhibited by tannic acid in the concentration range 0–200 M. However, insulin-stimulated autophosphorylation of the insulin receptor, and receptor-associated tyrosine kinase phosphorylation of RR-SRC peptide, were inhibited by tannic acid at concentrations as low as 25 M. Our data do not support the current speculation that tannins affect the activity of peptide hormones by binding to them. Therefore, our finding opens up a new perspective in the understanding of the mode of action of tannins on such hormones.     

Alkaline phosphatase in human lymphocyte subpopulations.

The levels of membrane alkaline phosphatase have been measured on different lymphocyte fractions from human peripheral blood separated on bovine serum albumin discontinuous gradients. The peak in enzyme activity was observed in a non-T-, non-B-cell fraction, rich in "null" lymphocytes; the lowest values were found in the fraction with the highest proportion of T-cells.     

Binding of centchroman--a nonsteroidal antifertility agent to human plasma proteins

Centchroman, a non-steroidal antifertility agent showed a low affinity (Kd = 13.19 X 10(-6) M) and nonsaturable binding to human plasma. Centchroman did not compete either with sex hormone binding globulin or corticosteroid binding globulin. Polyacrylamide gel electrophoresis and temperature dependent binding characteristics revealed that the protein responsible for centchroman binding to human plasma resembles albumin.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B3 to bovine serum albumin. Aflatoxin B3 was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B1 (AFB1) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B1, B2, G1, and G2 when tritiated AFB1 was used as the marker ligand. The concentrations causing 50% inhibition of binding of 3H-AFB1 to the antibodies by unlabeled aflatoxins B1, B2, G1, G2 and B3 were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B1 and G1, two of the most important toxic metabolites produced by Aspergillus flavus and A. parasiticus.     

Sur l'interaction entre aldéhydes ou réductones et acides aminés ou protéines

Summary Proteins were exposed to acetaldehyde vapour or mixed with glucose in solution and the basic groups determined by their dy binding capacity. Only serum albumin shows a marked decrease of basic groups and of the carbon/nitrogen ratio as a sign of condensation of aldehyde and amine groups. These results were corroborated by the direct estimation of the aldehyde resine formed on the protein: serum albumin and fibrin treated with acetaldehyde vapour contained 5 to 10 times more resin than the other proteins examined. No rapid combination between serum proteins and glucose could be demonstrated by the dy method or by the kinetics of change of pH. The slow reaction seems not to be dependent on denaturation.     

Binding of centchroman—a nonsteroidal antifertility agent to human plasma proteins

Summary Centchroman, a non-steroidal antifertility agent showed a low affinity (K_d=13.19×10^–6 M) and nonsaturable binding to human plasma. Centchroman did not complete either with sex hormone binding globulin or corticosteroid binding globulin. Polyacrylamide gel electrophoresis and temperature dependent binding characteristics revealed that the protein responsible for centchroman binding to human plasma resembles albumin.Acknowledgment. The authors are grateful to Dr. Nitya Nand for his interest in this investigation. CDRI Communication No. 3333.     

9,10-Dihydroergotamine: Production of antibodies and radioimmunoassay

Summary Antibodies against 9,10-dihydroergotamine (DHE) were produced by immunizing rabbits with a conjugate of 6-nor-6-carboxymethyl-9,10-dihydroergotamine and bovine serum albumin. A highly specific and sensitive radioimmunoassay for DHE has been developed.We are greatly indebted to Dr.T. Fehr for the synthesis of 6-nor-6-carboxymethyl-9,10-dihydroergotamine, and to Dr.E. Schreier for providing us with 9,10-dihydroergotamine-[13-³H]-base of high specific activity.     

On the binding of steroid sulfates to albumin

Summary ³H-Labeled steroid sulfates, sulfate of estrone (E₁S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E₁S). The apparent equilibrium constants (K) of the tracers did not measuraby change at concentrations of the non-radioactive sulfates below 10^–5 mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of³H-E₁S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E₁S in vivo, because of its preferetial occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.Acknowledgment. This investigation received financial support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organisation, and from the Ford Foundation.     

The dopaminergic innervation as observed by immunohistochemistry using anti-dopamine serum in the rat cerebral cortex

Summary By using an antiserum raised against dopamine bound to bovine serum albumin, thinner dopamine-labeled nerve terminals were visualized immunohistochemically within neocortical areas, in addition to well-documented dopaminergic innervation into the prefrontal and limbic cortices.This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas, Japanese Ministry of Education, Science, Culture (No. 62623002).     

Stabilisierung der oxydativen Phosphorylierung isolierter Mitochondrien durch artspezifische und artunspezifische Serumproteine

Summary Addition of 3 mg bovine serum albumin/mg mitochondrial-N enhances and stabilizes the phosphorylation capacity of isolated beef liver mitochondria. Albumin reduces the fluctation of the resulting P/O-rates and brings about a better reproducibility of the entire method. The same effect is produced by addition of bovine -globulin in half the concentration of albumin, while the effect of heterologous proteins (e. g. human albumin) is inferior.     

Production of active and passive anaphylactic shock in the WBB6F1 mouse, a mast cell-deficient strain

The role of mast cells in active and passive anaphylactic shock was examined using the WBB6F1 mouse, a genetically mast cell-deficient strain. Lethal anaphylactic shock occurred at high incidence rates in mice actively sensitized to bovine serum albumin (BSA). The reaction was specific to BSA since the shock could not be elicited by human or guinea pig serum albumin in these animals. Lethal shock could be prevented by CV-3988 but not by cyproheptadine, which suggests that the shock is mediated by PAF but not by histamine and serotonin. Similarly, lethal shock was provoked by homologous antigens in mice which had been passively sensitized with allogeneic anti-benzylpenicilloyl (BPO) IgG1 monoclonal antibody or with allogeneic or xenogeneic anti-BSA antiserum, but not in those sensitized with allogeneic anti-BPO IgE monoclonal antibody. These findings suggest that mast cells are not necessarily required for anaphylactic shock in the mouse.     

Immunization of female rabbits against testosterone stimulates testosterone accumulation by isolated ovarian follicles

Summary Ovarian follicles isolated from female rabbits after active immunization against testosterone-3-oxime bovine serum albumin produced more testosterone than similar follicles from controls.This work was supported by the Medical Research Council of Canada MT 4192.     

Transfer and induction of delayed hypersensitivity to methylated bovine serum albumin in the absence of adjuvant]

Delayed hypersensitivity to methylated bovine serum albumin may be induced in Mice without adjuvant, by injecting the antigen either with sensitized T lymphocytes, or by injecting just the antigen in Mice preptreated with Cyclophosphamid.     

Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum

Addition of epidermal growth factor to culture medium without calf serum suppressed the increase in cell volume and then enhanced the survival of BSC-1 cells attached to culture dishes. However, these effects of epidermal growth factor were not observed in the case of cells on dishes coated with heat-denatured bovine serum albumin.     

Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum

Summary Addition of epidermal growth factor to culture medium without calf serum suppressed the increase in cell volume and then enhanced the survival of BSC-1 cells attached to culture dishes. However, these effects of epidermal growth factor were not observed in the case of cells on dishes coated with heat-denatured bovine serum albumin.