title: Enhanced clearance of specifically bound digoxin from human myocardial and skeletal muscle samples by specific digoxin antibody fragments: subsequent complete digitalis glycoside receptor (Na,K-ATPase) quantification.
abstract: The effect of digoxin antibody fragments (Fab) on clearance of specifically bound digoxin from its specific receptor (Na, K-ATPase) was studied in human heart left ventricle (LV) and vastus lateralis skeletal muscle (SK) samples obtained postmortem. Initially, [3H]digoxin was bound to samples at conditions giving high relative occupancy of receptor. Half-life (t1/2) for its net release from LV in buffer was 32.2, 6.7, and 0.9 h at 0 degrees, 30 degrees, and 37 degrees C, respectively. For SK, t1/2 was 5.4 h in buffer at 30 degrees C. Inhibition of rebinding of digoxin by addition of specific digoxin Fab (5 x 10(-7) M) or excess unlabeled digoxin (1 x 10(-4) M) to buffer at 30 degrees C increased net release rate for specifically bound digoxin 2.5- to 3.0-fold in heart and SK. [3H]Digoxin was also bound to samples at conditions giving low relative occupancy. Samples were subsequently washed in buffer containing 5 x 10(-7) M specific digoxin Fab for 16 h at 30 degrees C. This wash reduced occupancy of receptors by digoxin from 10 to 0.5% in LV and from 9 to 0.3% in SK, respectively. At variance with wash at 37 degrees C, this procedure allowed subsequent vanadate-facilitated complete quantification of Na,K-ATPase by [3H]ouabain binding; values were 378 +/- 13 and 370 +/- 12 pmol/g wet weight (p greater than 0.6) in LV and 309 +/- 19 and 315 +/- 16 pmol/g wet weight (p greater than 0.7) in SK with and without previous wash, respectively (mean +/- SEM, n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Medicine B, Rigshospitalet, Copenhagen, Denmark.
authors.forename: T A
authors.initials: TA
authors.lastname: Schmidt
authors.forename: K
authors.initials: K
authors.lastname: Kjeldsen
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D004077:Digoxin / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006207:Half-Life; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D066298:In Vitro Techniques; D008297:Male; D009132:Muscles / Q000201:enzymology / Q000378:metabolism*; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism; D011955:Receptors, Drug / Q000032:analysis / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000378:metabolism*
chemical_list: D007128:Immunoglobulin Fragments; D011955:Receptors, Drug; C027082:digitalis receptor; D010042:Ouabain; D004077:Digoxin; D000254:Sodium-Potassium-Exchanging ATPase