Optimización de la determinación de ácidos biliares séricos por método enzimático fluorométrico

TRIPODI VP; VIZIOLI NM; VESCINA MC; MAMIANETTI A; GARRIDO D; CARDUCCI CN*

ACTA BIOQUíMICA CLíNICA LATINOAMERICANA

Federación Bioquímica de la Provincia de Buenos Aires

Lugar: La Plata; Año: 1996 vol. 30 p. 103 - 109

0325-2957

Solid-phase extraction of serum bile acids (SBA) previous precipitation of plas­ma proteins, was performed to improve the determination of 3a-hydroxy derivatives by enzymatic fluorometric assay. To precipitate proteins, cold acetonitrile was used and the supernatant was concentrated by evaporation and then diluted with aceto­nitrile: water (3:70). The organic layer was applied to a SPE-C18 column and the bi­le acids were subsecuently eluted with methanol. The eluent was evaporated to dry­ness, taken up in methanol and determined by'espectrofluorometric assay employing 3a-hydroxysteroid dehydrogenase, _-NAD, diaphorase, and resazurin. Performance of sample clean-up was' evaluated by labelling the serum with [24-14Cl glycocholic acid. The recovery was 89.0:!: 1.3 % (SDJ, RSD = 1.4 % (n=9, for 3 days). Serum bi­le acids from healthy people were analyzed by this method and a mean of2.61:!: 0.39 _ (SEM) (n=27) was obtained. This methodology improved selectivity of enzyma­tic assay, elimination of interferences and preconcentration of SBA previous to their disruption of protein binding.