RESUMEN
The 19F NMR spectrum of triflupromazine hydrochloride (TFZ) in a buffer solution (pH 6.8) showed a single sharp signal of the TFZ CF3 group at 13.5 ppm from the external trifluoroacetic acid. The addition of 1 mM HSA or BSA to the sample solution caused a split of the CF3 signal into two broadened signals shifted to slightly lower (0.2 ppm) and higher (0.7 ppm) fields, respectively, from the original position. Denaturation of the albumins by guanidine hydrochloride (3M) restored the two broadened signals to a slightly broadened single signal, indicating that TFZ has at least two binding sites on HSA and BSA, respectively. From the competitive binding 19F NMR experiments using Warfarin (Site-I ligand), l-tryptophan (Site-II ligand), NaCl, and oleate, the signal at high field was assigned to the TFZ bound to Site II. Comparison of the signal intensity revealed that the affinity of TFZ for Site II on HSA was considerably higher than that on BSA. The low-field signal could be identified as a weight-averaged signal between nonspecifically bound TFZ to HSA (BSA) and free TFZ in the water phase. In the presence of physiological concentrations of NaCl, major binding of TFZ to HSA and BSA was considered to be nonspecific. The present work indicates that 19F NMR is very useful for obtaining important detailed information regarding the binding of fluorinated drugs to serum albumins.
Asunto(s)
Antipsicóticos/sangre , Albúmina Sérica/análisis , Triflupromazina/sangre , Animales , Unión Competitiva/efectos de los fármacos , Bovinos , Humanos , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Unión Proteica , Albúmina Sérica Bovina/análisisRESUMEN
We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a C18 reversed-phase column and spectrophotometric detection. The standard curves for the drug and metabolite are linear up to 20.0 mg/L, with a lower limit of detection of 0.16 mg/L. The CVs for intra-assay precision were 5.0% at 0.58 mg/L and 2.9% at 5.96 mg/L; for inter-assay precision, they were 9.6% at 0.52 mg/L and 6.1% at 2.09 mg/L. Lipemia, hemoglobin, and bilirubin up to 300 mg/L do not interfere with this assay. None of > 550 cardiac patients' samples tested contained a compound that interferes with this assay.
Asunto(s)
Amiodarona/análogos & derivados , Amiodarona/sangre , Cromatografía Líquida de Alta Presión , Humanos , Concentración de Iones de Hidrógeno , Triflupromazina/sangreRESUMEN
Radioactively labeled 7-azido-fluphenazine and 7-azido-triflupromazine methiodide have been synthesized and their binding to membranes of intact red blood cells and to ghosts was compared after irradiation. The results indicated that tertiary phenothiazines react with integral membrane components. We conclude from the results that amphiphilic substances solubilize in biological membranes. This is in contradiction to the proposal that these compounds are excluded from the hydrophobic core of biological membranes (Conrad & Singer (1979) Proc.Natl.Acad.Sci.U.S.A. 76, 5202-5206 and (1981) Biochemistry 20, 808-818).