Study of isotope effects on protein binding by gas chromatography/mass spectrometry of theophylline-phenobarbitone and 2H, 13C, 15N isotopomers.
We describe a comparative study of human serum albumin (HSA) binding by equilibrium dialysis (pH 7.4, 37 degrees C, 3 h) for two groups of isotopic analogues: theophylline and 1-C(2H3)theophylline; unlabelled, 5(ethyl(2H5],-5(phenyl(2H5] and 1,3-15N;2-13C-phenobarbitone. Bound and free drug fractions are quantified by combined gas chromatography/mass spectrometry. In three instances, protein binding parameters are greatly affected by isotopic substitution, namely for: theophylline and 1-C(2H3)theophylline with isotope effects on total binding site concentration (N), affinity constant (Ka) and extent of HSA binding (%) respectively, equal to: NL/NH = 0.51; KaL/KaH = 1.78; %L/%H = 0.96 (L (light) and H (heavy) represent the unlabelled and labelled analogue respectively); phenobarbitone/-5-(phenyl(2H5]phenobarbitone, NL/NH = 1.72; KaL/KaH = 0.56; %L/%H = 1.26; phenobarbitone/1,3-15N;2-13C phenobarbitone, NL/NH = 2.95; KaL/KaH = 0.44; %L/%H = 1.32, together with a change from one (saturable) to two (saturable + non-saturable) families of albumin binding sites in the latter case. Contrasting with these data, no HSA binding isotope effect was observed on phenobarbitone C5 ethyl deuteration.
Cherrah
Y
Y
LEACM Faculty of Pharmacy, Lyon, France.
Falconnet
J B
JB
Desage
M
M
Brazier
J L
JL
Zini
R
R
Tillement
J P
JP
D002247:Carbon Isotopes; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D066298:In Vitro Techniques; D007554:Isotopes; D009587:Nitrogen Isotopes; D010634:Phenobarbital / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D013806:Theophylline / Q000378:metabolism*
D002247:Carbon Isotopes; D007554:Isotopes; D009587:Nitrogen Isotopes; D012709:Serum Albumin; D003903:Deuterium; D013806:Theophylline; D010634:Phenobarbital