The hepatic transport of hippuric acid (HA), a glycine-conjugated metabolite of benzoic acid that exhibits only modest plasma albumin binding (binding association constant of 2.1 × 103M−1), was studied in the single-pass perfused rat liver (12 ml/min), using the multiple indicator dilution (MID) technique. The venous recovery of [3H]HA on portal venous injection of a MID dose containing a mixture of a set of noneliminated reference indicators and [3H]HA revealed a survival fraction of unity, corroborating the lack of disappearance of bulk HA from plasma. When the outflow recovery was fitted to the barrier-limited model of Goresky et al. (C. A. Goresky, G. G. Bach, and B. E. Nadeau. J. Clin. Invest. 52: 991–1009, 1973), the derived influx ( P in S ) and efflux ( P out S ) permeability-surface area products were found to be dependent on the concentration of HA (1–930 μM); P in S and P out S were ∼3.5 times the plasma flow rate at low HA concentration, but decreased with increasing HA concentration. All values, however, greatly exceeded the expected contribution from passive diffusion, because the equilibrium distribution ratio of chloroform to buffer for HA was extremely low (0.0001 at pH 7.4). The tissue equilibrium partition coefficient ( P in/ P out, or ratio of influx to efflux rate constants, k 1/ k −1) was less than unity and decreased with concentration. The optimized apparent Michaelis-Menten constant and maximal velocity were 182 ± 60 μM and 12 ± 4 nmol ⋅ s−1 ⋅ g−1, respectively, for influx and 390 ± 190 μM and 29 ± 13 nmol ⋅ s−1 ⋅ g−1, respectively, for efflux. In the presence ofl-lactate (20 mM), however, P in S for the uptake of HA (174 ± 3 μM) was reduced. Benzoic acid (10–873 μM) was also effective in reducing hepatic uptake of HA (5.3 ± 0.9 μM). These interactions suggest that MCT2, the monocarboxylate transporter that mediates the hepatic uptake of lactate and other monocarboxylic acids, may be involved in HA transport.
Conjugative metabolism of 4-methylumbelliferone in the rat liver: Verification of the sequestration process in multiple indicator dilution experiments.