LOCALIZATION OF GLUTATHIONE CONJUGATION ACTIVITIES TOWARD BROMOSULFOPHTHALEIN IN PERFUSED-RAT-LIVER - STUDIES WITH THE MULTIPLE INDICATOR DILUTION TECHNIQUE

The plasma binding and conjugation kinetics of bromosulfophthalein (BS P) with glutathione (GSH) were studied in the single-pass in situ perf used rat liver (portal vein perfusion at 10 ml/min); GSH in post-mitoc hondrial fractions of the liver at the end of the experiment was exami ned 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 x 10(7) M-1, and one of 7.4 equiva lent sites of association constant, 1.3 x 10(5) M-1. Nonlinear binding was observed within between 5-1500 muM BSP. At varying input concentr ations (0.4-250 muM) of BSP, the unbound fraction was extremely low (< 0.005) and the hepatic extraction ratio declined from 0.67 to 0.15; lo ss of BSP was primarily caused by GSH conjugation to form BSP-GSH, whi ch appeared exclusively in bile. Additionally, unchanged BSP and two v ery minor unidentified metabolites were also excreted in bile. The for mation of BSP-GSH proceeded with an apparent V(max) of 22 nmol/min/g a nd a K(M) of 0.05 muM, whereas the parameters for BSP excretion were 0 .85 nmol/min/g and 0.02 muM, respectively. Within the concentration ra nge of BSP examined, GSH availability did not appear to be rate-limiti ng in the formation or excretion of BSP-GSH. Under conditions that ens ured first-order conditions (<2 muM), the method of hepatic artery-por tal vein/hepatic artery-hepatic vein (HAPV/HAHV) perfusion, with arter ial 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 e xamine the localization of GSH S-transferase activities. At steady-sta te, a dose containing multiple, nopeliminated indicators was injected into the HA for estimation of the accessible cellular water space duri ng 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 ra tios (HAHV/HAPV) to be made. HAHV/HAPV ratios for total (0.49, p < 0.0 5), metabolic 0.22, p < 0.05), and biliary (0.61, p > 0.05) removal ra te constants suggest that the GSH conjugation activity toward BSP is e nriched in the perihepatic venous region, whereas the biliary excretio n of BSP does not appear to be zonally directed.