Effects of perfusate flow rate on measured blood volume, disse space, intracellular water space, and drug extraction in the perfused rat liver preparation: Characterization by the multiple indicator dilution technique

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Abstract
The effect of hepatic blood flow on the elimination of several highly cleared substrates was studied in the once-through perfused rat liver preparation. A constant and low input concentration of ethanol (2.0 mM), [14C]-phenacetin and [3H]-acetaminophen (0.36 and 0.14 μM, respectively), or meperidine (8.1 μM) was delivered once-through the rat liver preparation in five flow periods (>35 min each); control flow periods at 12 ml/min were interrupted by flow changes to 8 or 16 ml/min. The steady-state hepatic availabilities (F or outflow survivals) at 12 ml/min were ethanol, 0.075±0.038; [14C]-phenacetin, 0.15±0.059; [3 H]-acetaminophen, 0.34±0.051; meperidine, 0.047±0.017. Flow-induced changes were different among the compounds: with reduced flow (8 ml/min), F was decreased for ethanol (0.061 ±0.032) and [3H]-acetaminophen (0.28±0.051), as expected, but was increased for [14C]-phenacetin (0.20 ±0.068) and meperidine (0.05 ±0.03); with an elevation of flow (to 16 ml/min), F was increased for all compounds, as expected of shorter sojourn times: ethanol, 0.13 ±0.065; [14C]-phenacetin, 0.22 ±0.062; [3H]-acetaminophen, 0.43 ±0.063; meperidine, 0.055±0.022. A marked increase in F for ethanol had occurred when flow changed from 12 to 16 ml/min due to nonlinear metabolism; the latter was confirmed by a reduction in the extraction ratios at increasing concentrations (1.8 to 11.4mM); this condition was not present for the other compounds. In order to explain the observations, we used the multiple indicator dilution technique to investigate the flow-induced behaviors of tissue distribution spaces of vascular and intracellular references in the perfused rat liver preparation. After a rapid injection of noneliminated reference materials [51 Cr-labeled RBC (vascular marker),125I-labeled albumin, [14C]-sucrose (extracellular markers), and [3H]-H2O (cellular marker)] into the portal veins of livers perfused at the randomly chosen flow rates (5, 8, 10, 12, 14, or 16 ml/min), the hepatic venous outflow profiles were characterized. Estimated sinusoidal blood volume, total albumin and sucrose distribution spaces, the Disse space, total water space, and the transit time for intracellular water showed strong correlations with blood flow rate. No correlation was found, however, between blood/water flow rate and intracellular water space (a space also accessed by substrates). At < 0.75 ml blood/min/g liver, intracellular water space was decreased, but at > 0.75 ml blood/min/g liver, the observed values were constant (0.635±0.024 ml/g liver) and independent of flow rate. Estimations of the mean transit time for cell water enabled calculations of sequestration rate constants (intrinsic clearance per ml cell water). The estimated sequestration rate constants for meperidine and phenacetin were decreased to 64% when flow was decreased from 12 to 8 ml/min, whereas those for acetaminophen (preformed or generated from phenacetin) were decreased minimally (10 to 11%), and these were generally unchanged for most compounds when flow was altered from 12 to 16 ml/min. The composite findings suggest that a critical flow is required to maintain maximal and constant accessibility into hepatocytes. Flow rates below this critical value affect hepatocyte recruitment differentially, as suggested by drug metabolic data. Below the critical flow rate, the reduction in intracellular space affected mostly metabolic processing of drugs that are mediated by enzymes located in the perihepatic venular region, but the effects are virtually imperceptible for biotransformation of drugs that involve enzyme systems in the periportal region.

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