In vitro evidence that urine composition affects the fraction of active furosemide in the nephrotic syndrome.
Diuretic resistance to furosemide in the nephrotic syndrome (NS) may result from binding of drug to filtered albumin within the renal tubule. In buffer solutions intended to partially mimic the luminal environment of the distal nephron during the NS, we examined several chemical properties to determine their effect on furosemide-albumin binding equilibria. Dissociation constants were obtained by measuring furosemide's quenching of human serum albumin's intrinsic tryptophan fluorescence over ranges of pH, ionic strength (IS) and osmolarity. Neither pH nor osmolarity significantly affected binding; however, incremental increases in IS between 0.0 and 1.0 produced increases in Kd from 0.65 +/- 0.05 to 34.38 +/- 1.72 microM, resulting in a 5- and 28-fold increase in the unbound furosemide fraction when the furosemide-albumin concentrations were 3.0:5.0 and 10.0:45.0 microM, respectively. Our results indicate that human serum albumin contains one high affinity binding site for furosemide that is sensitive to IS. Because of changes in the concentrations of reactants as well as IS that can occur in nephron segments distal to furosemide's site of action, we conclude that the amount of unbound (i.e., pharmacologically active) drug in voided urine will not necessarily correspond to the amount at the active site. To clinically assess the pharmacodynamic consequence of protein binding in the NS, changes in the concentration of the reactants and IS in the distal nephron must be minimized so that the unbound furosemide measured in voided urine will accurately reflect the amount at the drug's active site.
Department of Medicine, Indiana University School of Medicine, Indianapolis.
J R
JR
Voelker
D M
DM
Jameson
D C
DC
Brater
D005665:Furosemide / Q000378:metabolism / Q000493:pharmacokinetics*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009404:Nephrotic Syndrome / Q000188:drug therapy*; D009994:Osmolar Concentration; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism; D014556:Urine / Q000502:physiology*
D012709:Serum Albumin; D005665:Furosemide