Localization of glutathione conjugation activities toward bromosulfophthalein in perfused rat liver. Studies with the multiple indicator dilution technique
Zhao, Y.; Snel, C.A.; Mulder, G.J.; Pang, K.S.
Drug Metabolism and Disposition the Biological Fate of Chemicals 21(6): 1070-1078
1993
ISSN/ISBN: 0090-9556 PMID: 7905386 Document Number: 415276
The plasma binding and conjugation kinetics of bromosulfophthalein (BSP) with glutathione (GSH) were studied in the single-pass in situ perfused rat liver (portal vein perfusion at 10 ml/min); GSH in post-mitochondrial fractions of the liver at the end of the experiment was examined as a potential rate-determining factor. BSP was highly bound to 1% albumin with at least two classes of binding sites: one of 0.17 site with an association constant of 1.9 times 10-7 M-1, and one of 7.4 equivalent sites of association constant, 1.3 times 10-5 M-1. Nonlinear binding was observed within between 5- 1500 mu-M BSP. At varying input concentrations (0.4-250 mu-M) of BSP, the unbound fraction was extremely low ( lt 0.005) and the hepatic extraction ratio declined from 0.67 to 0.15; loss of BSP was primarily caused by GSH conjugation to form BSP-GSH, which appeared exclusively in bile. Additionally, unchanged BSP and two very minor unidentified metabolites were also excreted in bile. The formation of BSP-GSH proceeded with an apparent V-max of 22 nmol/min/g and a K-M of 0.05 mu-M, whereas the parameters for BSP excretion were 0.85 nmol/min/g and 0.02 mu-M, respectively. Within the concentration range of BSP examined, GSH availability did not appear to be rate-limiting in the formation or excretion of BSP-GSH. Under conditions that ensured first-order conditions ( lt 2 mu-M), the method of hepatic artery-portal vein/hepatic artery-hepatic vein (HAPV/HAHV) perfusion, with arterial delivery of BSP (HA, 2 ml/min) and blank perfusate entering either into the PV or HV (10 ml/min; total flow was 12 ml/min) was used to examine the localization of GSH S-transferase activities. At steady-state, a dose containing multiple, noneliminated indicators was injected into the HA for estimation of the accessible cellular water space during HAPV and HAHV. Values of the extraction ratio of BSP for HAPV (0.42 +- 0.06) and HAHV (0.09 +- 0.06) and excretion rates of BSP-GSH and BSP allowed estimation of periportal activity/whole liver activity ratios (HAHV/HAPV) to be made. HAHV/HAPV ratios for total (0.49, p lt 0.05), metabolic 0.22, p lt 0.05), and biliary (0.61, p gt 0.05) removal rate constants suggest that the GSH conjugation activity toward BSP is enriched in the perihepatic venous region, whereas the biliary excretion of BSP does not appear to be zonally directed.