The Binding of Furosemide to Serum Proteins in Elderly Patients: Displacing Effect of Phenprocoumon

Abstract: The percentual binding of furosemide (5 μg/ml) was slightly but significantly lower in serum from elderly patients than in serum from normal blood donors (96.5±0.7 versus 97.7±0.3). A significant positive correlation was demonstrated between protein binding and albumin concentration in serum. The reduced binding in the elderly could be explained by an observed decrease in the concentration of albumin in the elderly. The percentual binding did not change in samples obtained during the first hour after the intravenous administration of 40 mg furosemide. Of 4-chloro-5-sulfamoyl antranilic acid (CSA) and antranilic acid (A) about 60% were bound to serum proteins in vitro and none of these two compounds affected the protein binding of furosemide. The addition of phenprocoumon (PPC) caused significant decreases in the percentual binding of furosemide in serum (5 μg/ml) at PPC values of 10 μg/ml and more. The effect of PPC on the binding of furosemide was studied in vivo in 7 patients receiving 40 mg furosemide intravenously without and with the simultaneous intravenous administration of PPC (3 mg per 10 kg body weight). In good accordance with the in vitro experiments the modest average decrease in furosemide binding, caused by concentrations of PPC not exceeding 6.2 μg/ml, was not significant.     

The binding of phenprocoumon to human plasma proteins

By in vitro experiments with equilibrium dialysis and 5 micrograms PPC per ml an average of 99.7 +/- 0.2% was bound to serum proteins. The binding in a solution with the albumin concentration which was present in the serum samples did not differ from this binding or from the binding in plasma where coagulation was prevented with either heparin or citrate. The binding constant in albumin solutions at 37 degrees was 4.5 +/- 0.31/mol x 10(-5) and an average of 1.16 +/- 0.04 primary binding sites was found. The association constant for the PPC albumin interaction was temperature dependent and the results of thermodynamic calculations suggested a combined ionic and non-polar type of binding as the change in enthalpy contributed with 40% of change in free energy. A large positive entropy change was found (15.79 Kcal/mol/degree K). The addition of phytomenadione to albumin solutions and of menadione to plasma caused a considerable decrease in the protein bound fraction of PPC, indicating the relevance of a study concerning the possible clinical consequences of these interactions.     

Binding of Sulfinpyrazone and its Metabolites in Human Serum and in Solutions of Human Serum Albumin

Abstract: The binding of sulfinpyrazone, its sulfone metabolite and its sulfide metabolite to serum protein was studied by equilibrium dialysis. At 20 μg/ml 99.1% of the parent compound was bound in serum, whereas 99.8% of the sulfide and 98.3% of the sulfone were bound at this concentration. The binding of the three compounds were studied in diluted serum and in solutions of human serum albumin (HSA). There was no evidence of binding to proteins other than albumin. The association constants to primary and secondary binding sites and the number of binding sites were calculated. For the sulfide a lower K₁-value in serum (0.76·10⁶ M^?1) than in the HSA solution (1.8·10⁶ M^?1) indicated the possible presence of a competitively bound substance in serum. In undiluted serum no displacing effect of the sulfide on sulfinpyrazone binding was found when both compounds were present in a concentration of 20 μg/ml, but in a HSA solution a pronounced sulfide induced displacement of the sulfinpyrazone from its primary binding site was shown. Acetylation of HSA depressed the binding of sulfinpyrazone but in undiluted serum there was no other effect on sulfinpyrazone binding by the addition of acetylsalicylic acid than could be explained by the displacing effect of salicylic acid. At concentrations at 20 μg/ml of sulfinpyrazone and above 50 μg/ml of the displacing agent significant displacement was demonstrated with phenylbutazone, tolbutamide and salicylic acid.     

Displacement by uricosuric agents of sodium urate bound to human serum albumin

The displacement of sodium urate by uricosuric agents from binding sites on human serum albumin (HSA) and normal serum has been demonstrated under physiological conditions in vitro (pH 7.4, ionic strength 0.16) over he oncentration range 1–13 mg/100 ml. At 37°, 0.5 mM sodium salicylate reduced urate bound to 5 g/100 ml of HSA from 19.6 to 9.7 per cent. At 22.5°, 22.6 per cent urate was bound to 5 g/100 ml of HSA, and this was reduced to 12.3 per cent by 0.5 mM salicylate, to 9.2 per cent by 0.5 mM phenylbutazone, to 14.6 per cent by 0.2 mM diflumidone, and to 17.9 per cent by 0.2 mM sulfaethidole. At 22.5°, pooled normal human serum (4.8 g/100 ml of albumin) bound 23.5 per cent urate; with 0.5 mM salicylate present 12.6 per cent urate was bound.     

Plasma protein binding of furosemide in the elderly

Summary The protein binding of furosemide was investigated in plasma from 22 old and 11 young subjects by equilibrium dialysis. The unbound fraction of furosemide was 3.16% in plasma from the elderly and 1.71% in plasma from the young. A significant correlation was found between the unbound fraction of furosemide and the plasma concentration of albumin. The average number of binding sites was 3.8 (elderly) and 2.7 (young) 10^–6 mol/g albumin. The average association constant (K) was 4.3 (elderly) and 4.2 (young) 10⁵ M^–1. By increasing the concentration of furosemide up to 200 µg/ml buffer the unbound fraction of the drug rose to 5.2% (elderly) and 3.5% (young).     

The effects of selected drugs on the in vitro protein binding of repaglinide in human plasma

This study investigated plasma protein binding by the novel oral hypoglycemic agent, repaglinide, and assessed the influence of other protein-bound drugs upon this process. Varying concentrations of [3H]-repaglinide (0.01 to 100 micrograms/ml) were incubated in solutions of plasma proteins (human serum albumin, HSA; alpha 1-acid glycoprotein, AAGP), or human plasma in the absence or presence of several test drugs. Protein binding was assessed using an ultrafiltration technique. At all concentrations tested, the mean binding of repaglinide in plasma was 98.5%, binding to HSA averaged 98.6%, and the binding to AAGP was saturable and remained below 50%. Warfarin 10 micrograms/ml, furosemide 0.2 microgram/ml, and tolbutamide 100 micrograms/ml, significantly reduced in vitro binding of repaglinide at 1 and 100 micrograms/ml versus control (p < 0.05), producing an 18-36% increase in free repaglinide. No reduction was found using 0.1 microgram/ml repaglinide. Diazepam, glibenclamide and nicardipine hydrochloride had no significant effects on the in vitro protein binding of repaglinide. These data suggest that the binding of repaglinide to HSA in human plasma has potential clinical significance, and that within the therapeutic range for repaglinide, the presence of the test drugs has no clinically relevant effects on repaglinide binding to plasma proteins.     

The Binding of Thiopental to Human Serum Albumin at Variable pH and Temperature

Abstract: The binding of thiopental was studied in vitro by equilibrium dialysis over the concentration range 0.8–80 μg/ml in solutions with 45 g albumin (HSA) per litre. The binding at 37° was from 79% to 67.6% at pH 6 while it was 94.5% to 93.0% at pH 9. At intermediate values of pH the observed values for binding were compatible with the assumption of a gradual transition of an N-form of HSA present exclusively at pH 6 to a B-form present exclusively at pH 9. The variation in binding could not be explained by changes in the balance between ionised and unionised drug (pK₂=7.6). An increase in r (number of thiopental molecules bound per HSA molecule) was demonstrated when the temperature was lowered to 2°. Graphs showing the relation between drug concentrations and percentual binding all showed a very modest slope and it was concluded that a traditional binding model with calculation of the number of binding sites and association constants was of little use in the evaluation of our binding studies of thiopental. The binding between thiopental and albumin may be characterized best by the solubility of thiopental in hydrofobic areas in the albumin solutions.     

Binding of two anthranilic acid derivatives to human albumin, erythrocytes, and lipoproteins: evidence for glafenic acid high affinity binding

The binding of two anthranilic acid derivatives, glafenic and floctafenic acids, to human erythrocytes and plasma proteins has been investigated in vitro by equilibrium dialysis. Despite their close chemical structures it was shown that the binding of the two compounds to serum albumin, lipoproteins, and erythrocytes was dramatically different both in quality and quantity. Using various techniques including fluorometry and circular dichroism, it was shown that glafenic acid binds to the human serum albumin (HSA) warfarin/azapropazone site and that floctafenic acid binds to both warfarin/azapropazone and benzodiazepine sites. Glafenic acid is strongly bound to HSA with n = 1, k = 2.4 X 10(6) liters/mol and to erythrocytes with N = 12.4 mumol/liter, K = 1.7 X 10(6) liters/mol. Floctafenic acid is bound with a weaker affinity to HSA, n = 2, k = 0.3 X 10(6) liters/mol and to erythrocytes, N = 2900 mumol/liter and K = 0.007 X 10(6) liters/mol.     

Distribution of chlorpromazine in a simplified blood influenced by various drugs

Summary The binding of chlorpromazine to erythrocytes and to albumin as influenced by other drugs was studied in a simplified blood (31.5±0.3% bovine erythrocytes, 4 g-% bovine serum albumin in 0.02 M phosphate buffer solution containing 0.15 M NaCl). the total concentration of chlorpromazine in the simplified blood was 10^–4 M, the concentration of the displacing drugs was 10^–3 M. After an incubation period of 3 h at 22° C the chlorpromazine concentration was determined in the albumin solution after centrifugation of the blood at 3000×g and in the aqueous phase after ultracentrifugation at 150000×g. Under control conditions 68.1±0.9% of chlorpromazine was bound to the erythrocytes, 28.5±0.9% was bound to albumin and 3.5±0.2% was free in the aqueous phase.The following substances were tested for their ability to alter the distribution of chlorpromazine in the simplified blood: Azetazolamide, amitriptyline, chlorimipramine, chlorothiazide, chlortetracycline, desoxycholic acid, diphenylhydantoin, imipramine, indomethacin, ioglycamic acid, oleic acid, oxytetracycline, phenindamine, phenprocoumon, phenylbutazone, probenecid, quinine sulfate, salicylic acid, stearic acid, sulfisoxazole, sulfamethoxypyridazine, suramin, tetracycline, thiopental. In most cases chlorpromazine shifted considerably between erythrocytes and albumin whereas the unbound fraction was not markedly changed.The effect of some of these drugs on the binding of chlorpromazine was also investigated in an albumin solution without erythrocytes. Evidence is presented that the binding capacity of albumin may be fundamentally changed when erythrocytes are present. It is concluded that binding studies merely with albumin solutions, plasma or serum may be misleading when pharmacokinetic implications are argued.Presented in part at the spring meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, 1972 (Krieglstein et al.).     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Influence of Fatty Acids on the Binding of Warfarin and Phenprocoumon to Human Serum Albumin with Relation to Anticoagulant Therapy

Warfarin and phenprocoumon binding to human serum albumin was studied by equilibrium dialysis. The first stoichiometric binding constant was 1.89 × 10⁵ M^?1 for warfarin and 2.40 × 10⁵ M^?1 for phenprocoumon. The affinity of warfarin was markedly increased on addition of up to 3 mol mol^?1 albumin of palmitic, stearic, oleic or linoleic acids with energetic couplings for co-binding of one molecule of each of the fatty acids and one molecule of warfarin of 0.9, 1.1, 0.7 and 0.6 kJ mol^?1, respectively. The affinity of phenprocoumon was only increased slightly on addition of palmitate with an energetic coupling of 0.3 kJ mol^?1. Six consecutive serum samples were obtained from each of 14 patients undergoing surgery. The serum affinity of the drugs varied considerably corresponding to free drug concentrations between 0.7 and 2.7% for warfarin and between 0.8 and 4.9% for phenprocoumon. The affinity of warfarin but not of phenprocoumon was correlated to the increasing plasma fatty acid concentration. Anticoagulant therapy with phenprocoumon may thus be less sensitive than warfarin to changes in the fatty acid concentration of plasma.     

Plasma protein binding and interaction studies with diflunisal,a new salicylate analgesic

The binding of diflunisal to human serum albumin and normal human plasma has been studied by equilibrium dialysis at 37°, pH 4. The plasma protein binding data were analysed according to a Scatchard model with two independent classes of binding sites. The number of binding sites and the corresponding association constants have been estimated by nonlinear least-squares regression analysis: N₁ = 2.1, k₁ = 5.28 × 10⁵M^?1, N₂ = 7.7 and K₂ = 0.17 × 10⁵M^?1. At a difluni concentration of 50 μg/ml on average 99.83 per cent of the drug was bound to plasma proteins. The in vitro plasma protein binding of diflunisal was impaired by salicylic acid and phenprocoumon, while diflunisal itself was displaced from its primary binding sites in plasma by salicylic acid and bilirubin. Tolbutamide had no effect on the binding of diflunisal to plasma proteins.     

The Biological Relevance of the Protein Binding of Diazoxide

Abstract: The biological importance of the binding of diazoxide to serum protein was studied in vitro. Contractions in the rings of the rabbit pulmonary artery evoked by electrical field stimulation or exogenous noradrenaline (NA) were studied in the absence and presence of serum albumin (45 g/1) in Krebs–Henseleit (K–H) solutions. Diazoxide concentrations up to 128 μg/ml in the K–H solution and to 256 μg/ml in albumin K–H solutions diminished the contractions and albumin reduced this diminishing effect. Because of an apparent binding of exogenous noradrenaline to albumin only the experiments with electrical field stimulation were used to assess the biological importance of the protein binding of diazoxide. At 2 μg/ml of diazoxide the free drug fraction determined by ultrafiltration was 5% but determined by our biological method the free (active) fraction was 65%. In accordance with a previous clinical study we conclude that the binding of diazoxide to serum protein is important for the transportation of the drug to the arteriolar wall which apparently has a much higher affinity for the drug than serum albumin     

Binding of chlorthalidone (Hygroton®) to blood components in man

Summary The binding of chlorthalidone to human blood components has been studied in vitro. The drug was preferentially taken up by red blood cells, the partition ratio between plasma and the cell fraction being dependent on the drug concentration. When the concentration of chlorthalidone in blood was less than 15–20 µg/ml, more than 98% of the compound was bound to red cells. Increasing the concentration resulted in an abrupt change of the partition ratio in favour of plasma, which indicates a saturable receptor for chlorthalidone in red cells, namely carbonic anhydrase (HCA). The association constant of the drug-enzyme complex K_assHCA was 2.76×10⁶ l/mole. For the two major isoenzymes of carbonic anhydrase, HCA-B and HCA-C, the association constants were different: K_assHCA-B=2.43×10⁶ l/mole and K_assHCA-C=5.69×10⁶ l/mole. The number of binding sites n=1 in all cases. In human serum at 37°C, over a concentration range of 0.02–7.7 µg/ml, 75.7% of chlorthalidone was bound to proteins. The major portion of the binding was to albumin (HSA), the association constant of the complex K_assHSA=1.18×10³ l/mole and the number of binding sites n=4. The much higher association constant of chlorthalidone with HCA than with HSA can account for selective uptake of the drug by red cells.     

The Determination of Naproxen by Spectrofluorometry and its Binding to Serum Proteins

Abstract A fluorometric method for the determination of naproxen in serum, albumin solutions and protein free buffer solutions is described. The detection limit is about 10 ng/ml. Furosemide, thiopental and salicylic acid did not show any disturbing fluorescence while phenprocoumon did. The in vitro binding of naproxen in albumin solutions and serum was studied by equilibrium dialysis. A small but significant increase was found in the percentual binding in albumin solutions as compared to serum. The percentual binding was not affected by changes in the pH from 5-8. By fitting the binding data to a model assuming two classes of binding sites, association constants and binding capacities were determined. A very high affinity and a high capacity were found. The association constant for the first class of binding sites was higher for human serum albumin than for Serum (8.5 versus 5.3 · 10⁶ M^-1). The difference in the protein binding to the first class of binding sites in human serum albumin and serum can be explained by the existence of a competitive inhibitor in serum.     

Heterologous serum albumin as tracer for plasma volume measurements evaluated in conscious nondehydrated and 48-hour water-deprived rats

The feasibility of using nonradioactive heterologous serum albumin measured by rocket immunoelectrophoresis as tracer for plasma volume measurements was evaluated by comparing simultaneous distribution volumes and plasma half-lives (t1/2) of human serum albumin (HSA) and Evans blue dye (T1824) in a group of conscious rats. The animals were studied under normal water intake and after a 48-h period of water deprivation. The kinetic behavior in plasma was qualitatively similar for both tracers, but T1824 distributed beyond the HSA space and had half-lives in plasma lower than those of HSA. Water deprivation induced a 14% decrease in the HSA space, from 2.7 +/- 0.1 to 2.4 +/- 0.2 ml/100 g body weight. We conclude that serum albumin spaces measured immunoelectrophoretically are a better reflection of true plasma volume than T1824 spaces and can be used advantageously in determinations of plasma volume.     

Protein binding of oxazepam and its glucuronide conjugates to human albumin

The binding of oxazepam and its glucuronide conjugates to human serum albumin (HSA), as well as the binding interactions of the drug and its metabolites, were examined by equilibrium dialysis and kinetic probe studies. Oxazepam and its S(+) glucuronide are bound to the HSA molecule with affinity constants of 3.5 X 10(5) M-1 and 5.5 X 10(4) M-1, respectively, which were independent of protein concentration over a range of 0.1 to 5.0 g/dl. The R(-) glucuronide bound weakly to albumin, with the binding parameter, N X K, increasing at lower albumin concentrations. Pre-acetylation of fatty acid free-HSA resulted in decreased binding of all three compounds, probably by altering the conformation of the binding sites. Kinetic probe studies with p-nitrophenyl acetate indicate that oxazepam and its S(+) glucuronide shared a common binding site on HSA, but that the R(-) glucuronide bound at another site. Oxazepam binding was unaffected by the presence of its glucuronide conjugates but was inhibited by fatty acids. The percentage of oxazepam bound to plasma proteins in patients with renal impairment (94%) was lower than in normal volunteers (97%). This lower binding can neither be attributed to lower albumin concentrations because of the large binding capacity of the protein and linearity of N X K nor to displacement by elevated concentrations of glucuronide conjugates, but it may be ascribed partly to increased plasma fatty acids.     

Binding of prazosin and propranolol at variable alpha 1-acid glycoprotein and albumin concentrations.

1. The effect of variable alpha 1-acid glycoprotein (AAG) and albumin (HSA) concentrations on the binding of prazosin and propranolol was assessed in plasma after surgery and in mixtures of AAG/HSA with concentrations mimicking those found in vivo. 2. On the pre-operative day the binding of prazosin and propranolol was 94.8% and 89.0%, respectively and 97.3% and 93.2%, respectively, 5 days after surgery. 3. In solutions containing mixtures of highly purified AAG and HSA representing the pre-operative state, 88.6% and 83.9% binding of prazosin and propranolol was observed, whereas for solutions mimicking post-operative plasma, the equivalent values were 94.6% and 91.4%, respectively. 4. The ratios between bound and unbound concentrations of both drugs were closely correlated to the concentrations of AAG, but not to the concentrations of HSA. 5. The present study demonstrates that AAG is responsible for the binding variability of prazosin and propranolol in plasma from post-operative patients.     

Untersuchungen zur Eiweißbindung durch kontinuierliche Ultrafiltration, 2. Mitt. Eiweißbindung von Warfarin

Protein Binding of Drugs Determined by Continuous Ultrafiltration, II: Protein Binding of Warfarin The binding of warfarin to human serum albumin has been investigated in the ranges (c_total) of 0.6–4.2 μg · ml^?1 (fluorescence detection) and 100–400 μg · ml^?1 (ultraviolet detection). The amount of unbound drug is 0.5% and nearly constant up to 100 μg · ml^?1. The other parameters, too, correspond fairly well to those obtained with other methods. There is no significant difference between the binding parameters of warfarin and phenprocoumon in therapeutically achieved concentrations.