Application of d-Penicillamine2,5-enkephalin to characterization of sandwich-cultured hepatocytes transport activities

Abstract

Abstract: AIM: To develop a sensitive and rapid method for determining the concentration of d-Penicillamine2,5-enkephalin (DPDPE) in hepatocytes and Hank's buffer by LC/MS/MS. And to evaluate the function of transporters in sandwich-cultured rat hepatocytes (SCRH) using DPDPE as the probe substrate.METHODS: Co-incubate DPDPE with SCRH treated by alpha-naphthylisothiocyanate, the model compound, and measure the accumulation and the efflux of DPDPE by SCRH. The concentration of DPDPE in hepatocytes or Hank's buffer was determined by LC/MS/MS.RESULTS: DPDPE had a good linearity from 0.5 to 50 ng/mL (r²=0.9995) in Hank's buffer and from 0.1 to 5 ng/well (r²=0.9997) in hepatocytes in the LC/MS/MS method established in this study. This method was sensitive enough to quantify DPDPE used as probe substrate in SCRH transport studies. The inter- and intra-day precision (RSD) were less than 15% and the recoveries were above 65%. The basolateral uptake and canalicular efflux of DPDPE by SCRH was inhibited by ANIT without change in the basolateral efflux.CONCLUSION: The analytical method established here is selective, sensitive and feasible to determine DPDPE used as probe substrate in the evaluation of membrane transporters in SCRH.

Key words: Transporter, Sandwich-cultured rat hepatocytes, LC/MS/MS, DPDPE

CLC Number:

R969.1

GUO Cen, HE Lei, YAO Dan, PAN Guo-yu, WANG Guang-ji. Application of d-Penicillamine2,5-enkephalin to characterization of sandwich-cultured hepatocytes transport activities[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2013, 18(5): 506-512.

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