S. Jacobsen

Variable binding of propranolol in human serum.

Human serum proteins were fractionated by ultracentrifugation and gel filtration. Binding of propranolol was determined by equilibrium dialysis. Propranolol was distributed to lipoproteins independent of drug concentration. Two groups of propranolol binding sites were found to be present in the protein preparation containing albumin, α1-acid glycoprotein, transferrin and prealbumin. The first binding site with a dissociation constant of 7.5 × 10−7 was present in number equivalent to concentration of α1-acid glycoprotein. The propranolol binding to serum samples from 21 healthy males expressed as binding ratio B/F and per cent binding ranged from 7.5 to 19.2 and 88.2 to 95.0 respectively. The binding ratio was correlated to concentration of α1-acid glycoprotein (r = 0.85, P < 0.001), but not to concentrations of albumin and lipoproteins. The results indicate that α1-acid glycoprotein is the main propranolol binding protein in human serum.

Effect of urine pH and flow on renal clearance of methotrexate

SummaryHydration and urinary alkalinization are used with high doses of methotrexate (MTX) to prevent precipitation of the drug in the renal tubules and consequential nephrotoxicity. The quantitative effect of these measures on the renal clearance of MTX was studied in 8 patients with normal renal function, and in 3 patients with reduced renal function. Multiple regression analysis indicated an influence of both factors on the ratio of the renal clearances of MTX and creatinine. In the eleven patients there was a linear correlation between this ratio and urine pH (p<0.001); the ratio increased from 0.88 at pH 5.5 to 2.62 at pH 8.4. The pH effect on this ratio was similar in the patients with normal and reduced kidney function. An increase in urine flow did not significantly increase the ratio between renal clearance of MTX and creatinine. The effect of urinary alkalinization on renal MTX clearance could be clinically exploited in patients with delayed elimination of MTX. The probable modifying effect of alkalinization of urine on the intentionally high plasma concentration after high dose MTX infusions should be further evaluated, particularly in patients with normal renal function.

Binding of quinidine in sera with different levels of triglycerides, cholesterol, and orosomucoid protein

Abstract Serum binding of quinidine was determined in vitro by equilibrium dialysis in sera from twenty-five healthy individuals. The sera had different levels of triglycerides, cholesterol and orosomucoid ( α 1 -acid glycoprotein), but with small variations in serum albumin concentration. Binding ratio (bound/free) and per cent binding varied from 2.0 to 5.4 and from 67.1 to 84.3% respectively. Binding ratios were linearly related to serum concentration of triglycerides ( r = 0.437, P r = 0.400 P r = 0.841, P r = 0.765, P r = 0.465, P r = 0.753, P

The binding of quinidine to protein fractions of normal human sera

Abstract In contrast to previous assumptions, albumin is not the only protein in normal human serum responsible for binding quinidine. Human serum proteins were fractionated by gel filtration and floatation. Quinidine binding was determined by equilibrium dialysis. The binding to low (LDL) and high (HDL) density lipoproteins exhibited two binding sites on each protein, and the dissociation constants K and number of binding sites n were calculated. LDL: K 1 = 2 × 10 −5 , n 1 = 1 and K 2 = 5·2 × 10 −4 , n 2 = 97. HDL: K 1 = 1·9 × 10 −5 , n 1 = 0·1 and K 2 = 1·1 × 10 −3 , n 2 = 14·7.

Effect of heparin and fatty acids on the binding of quinidine and warfarin in plasma

Abstract After an intravenous injection of heparin, the plasma protein binding of warfarin was greatly increased while the binding of quinidine seemed to be less affected. The increased binding of warfarin seemed partly due to the release of plasma non-esterified fatty acids, NEFA, but the level of NEFA alone could not explain the interindividual variations of plasma warfarin binding. Addition in vitro of palmitic acid to serum demonstrated an increased binding of warfarin and an unaltered binding of quinidine up to a serum NEFA level of 3.0 meq/l. Albumin isolated from post heparin plasma revealed an increased binding affinity for warfarin at the warfarin high affinity binding sites, and a slightly depressed affinity for quinidine. The low affinity binding sites on the albumin molecule for both drugs did not seem to be influenced by NEFA.

Elevated level of β-adrenergic receptors in hepatocytes from regenerating rat liver: Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and β-adrenergic receptors in cultured hepatocytes: Dependence on protein and RNA synthesis and suppression by isoproterenol

Adult male rat hepatocytes, which normally respond poorly to β‐adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine‐sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the β‐adrenoceptor ligand [125I]cyanopindolol. The emergence of β‐adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydortestosterone to the cultures, singly or in combination, did not prevent the augmented β‐adrenergic responsiveness. The increase in catecholamine‐sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3‐hourly intervals led to a dose‐dependent suppression of the rise in isoproterenol‐responsive adenylate cyclase and prevented the increase in β‐adrenoceptor binding.Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.

Pharmacokinetics of quinidine related to plasma protein binding in man.

SummaryThe disposition and plasma protein binding of quinidine after intravenous administration were studied in 13 healthy subjects. Plasma protein binding, expressed as the fraction of quinidine unbound ranged from 0.134–0.303 (mean 0.221). Elimination rate constant (β) varied from 0.071 to 0.146 h−1 (mean 0.113), and apparent volume of distribution (Vβ) varied from 1.39–3.20 l · kg−1β (mean 2.27). Total body clearance was 2.32–6.49 ml min−1 · kg−1. There was a positive linear correlation between the plasma fraction of unbound quinidine and both Vβ (r=0.885, p<0.01) and total body clearance (r=0.668, p<0.05). No significant correlation existed between the fraction of unbound quinidine in plasma and the elimination rate constant. The results show that both the apparent volume of distribution and total body clearance of quinidine are proportional to the unbound fraction in plasma. This implies that the total plasma concentration of quinidine at steady state will change with alterations in plasma binding, whilst the concentration of unbound compund and its elimination rate will remain unaffected.

Increased binding of quinidine to serum albumin and lipoproteins in anuric rats

The purpose of this work was to identify the main quinidine binding molecules of rat serum and those macromolecules responsible for increased quinidine binding in serum from rats with acute anuria. Rat sera were fractionated by gel filtration, ultracentrifugation and anion exchange chromatography. The binding of quinidine to sera and serum fractions was determined by equilibrium dialysis. The experiments demonstrated that albumin and lipoproteins are the main quinidine binding molecules in serum from normal and anuric rats. The increased binding by serum from anuric rats is due to both serum lipoproteins and albumin, even though the concentration of albumin is decreased from 2.8 to 1.7 g/100 ml. The increased binding to albumin may be due to conformation changes induced by endogenous substances. The increased binding to lipoproteins may be caused by an increased concentration of pre-beta-lipoprotein.

Down-regulation of surface beta-adrenoceptors on intact human mononuclear leukocytes: Time-course and isoproterenol concentration dependence

Incubation of human mononuclear leukocytes (MNL) in vitro with isoproterenol resulted in a rapid loss of surface beta-adrenoceptors, determined by radioligand binding at 4 degrees. Isoproterenol concentrations in the range of 10 nM to 100 microM resulted in significant down-regulation of beta-adrenoceptors. At a concentration of 1 microM isoproterenol, the time-dependent loss of surface beta-adrenoceptors closely paralleled the loss in isoproterenol-stimulated adenylate cyclase activity. If receptor number in intact cells was determined at 32 degrees, hardly any loss in receptor number was observed, due to reversal of down-regulation during the incubation period. When beta-adrenoceptor number in broken cell preparations was determined by [125I]cyanopindolol binding at 37 degrees no significant loss was observed, even after 2 hr of isoproterenol treatment, while [3H]CGP-12177 binding resulted in a similar reduction in binding sites as in intact cells. Reversal of loss in surface beta-adrenoceptors was rapid after 1 hr pretreatment with isoproterenol, but followed a biphasic time course after 4 hr pretreatment, with an initial rapid return of about 40% of the down-regulated receptors, followed by a slow, gradual reappearance of receptors. The results indicate that catecholamine exposure leads to a rapid sequestration of MNL surface beta-adrenoceptors away from the cell surface, to a compartment where they are inaccessible to the hydrophilic ligand [3H]CGP-12177 as well as to the lipophilic ligand [125I]cyanopindolol at 4 degrees. Up to 2 hr of isoproterenol treatment does not lead to any breakdown of sequestered beta-adrenoceptors, as they are still recognized by [125I]cyanopindolol binding in broken cell preparations.