[Determination of total plasma homocysteine and other aminothiols by liquid chromatography coupled to the detection by fluorescence].
Plasma concentrations of homocysteine, cysteine, cysteinylglycine and glutathione were measured using a HPLC technique with fluorescence detection of the derivatives obtained with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F). Blood was drawn into chilled EDTA-evacuated tubes. After centrifugation at 4 degrees C without delay, plasma samples were kept frozen at -20 degrees C until analysis. Reduction of protein bound aminothiols and disulfides standards was achieved with tri-n-butylphosphine. N-acetylcysteine was used as internal standard. After protein precipitation, derivatization was carried out at pH 8.0 and 50 degrees C for 20 min. Stability of ABD-thiols was ensured for at least 5 days by lowering pH to 2. Derivatives were separated by isocratic elution on a Waters mu Bondapak C18 column (10 microns, 3.9 x 300 mm) with 0.1 M phosphate buffer pH 3.2 containing 10% acetonitrile. Excitation and emission wavelengths were 385 and 515 nm. Retention times were 4.9, 5.8, 7.3, 9.9 and 20.1 min respectively for cysteine, cysteinylglycine, homocysteine, glutathione and N-acetylcysteine. Peaks were quantified by comparison to a standard curve prepared by plotting peak height versus the different levels of known standard solutions after normalization with internal standard. Between-run CVs varied from 5 to 8.5%. The detection limit was < 0.5 mumol/l for homocysteine and glutathione. In plasma samples from healthy subjects, concentration of homocysteine was higher in men than in women (11.0 +/- 2.9 versus 9.2 +/- 2.7 mumol/l, p < 0.01). These values are similar to those obtained with other widely used methods.
Jacob
N
N
Laboratoire de biochimie C, Groupe hospitalier Pitié-Salpêtrière, Paris.
Guillaume
L
L
Garçon
L
L
Foglietti
M J
MJ
D000111:Acetylcysteine / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000097:blood; D004151:Dipeptides / Q000097:blood; D005260:Female; D005453:Fluorescence; D005978:Glutathione / Q000097:blood; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D008297:Male; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000097:blood*
D004151:Dipeptides; D013438:Sulfhydryl Compounds; D006710:Homocysteine; C028505:cysteinylglycine; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine
Dosage de l'homocystéine plasmatique totale et autres aminothiols par chromatographie liquide couplée à la détection par fluorescence.