K. Sandy Pang

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

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.

A review of metabolite kinetics

The importance of metabolites as active and toxic entities in drug therapy evokes the need for an examination of metabolite kinetics after drug administration. In the present review, emphasis is placed on single-compartmental characteristics for a drug and its primary metabolites under linear kinetic conditions. The determination of the first-order elimination rate constants for drug and metabolite are also detailed. For any ithprimary metabolite miformed solely in liver, kinetic parameters with respect to primary metabolite formation under first-order conditions require a comparison of the areas under the metabolite concentration-time curve after drug and preformed metabolite administrations. These area ratios hold regardless of the number of noneliminating compartments for the drug and metabolite. These parameters include fmiand gmi,the fractions of total body clearance that respectively furnishes mito the general circulation and forms mi,and hmi,the fraction of hepatic clearance responsible for the formation of mi.Moreover, the fraction of dose dmiconverted to form miis defined with respect to the route of drug administration. The inherent assumption of these estimates, however, requires that the extent of sequential elimination of the generated mibe identical to the extent of metabolism of preformed mi.Discrepancies have been found, and may be attributed mostly to the uneven distribution of drug-metabolizing activities as well as to the presence of diffusional barriers. Other linear systems that involve miformation from multiple organs are briefly described.

Hepatic modeling of metabolite kinetics in sequential and parallel pathways: Salicylamide and gentisamide metabolism in perfused rat liver

Previous data on salicylamide (SAM) metabolism in the perfused rat liver had indicated that SAM was metabolized by three parallel (competing) pathways: sulfation, glucuronidation, and hydroxylation, whereas sequential metabolism of the hydroxylated metabolite, gentisamide (GAM), was solely via 5-glucuronidation to form GAM-5G. However, under comparable conditions, preformed GAM formed mainly two monosulfate conjugates at the 2- and 5-positions (GAM-2S and GAM-5S); 5-glucuronidation was a minor pathway. In the present study, the techniques of normal (N) and retrograde (R) rat liver perfusion with SAM and mathematic modeling on SAM and GAM metabolism were used to explore the role of enzymic distributions in determining the dissimilar fates of GAM, as a generated metabolite of SAM or as preformed GAM. Changes in the steady-state extraction ratio of SAM (E) and metabolite formation ratios between N and R perfusions were used as indices of the uneven distribution of enzyme activities. Two SAM concentrations (134 and 295 μM) were used for single-pass perfusion: the lower SAM concentration exceeded the apparent Kmfor SAM sulfation but was less than those for SAM glucuronidation and hydroxylation; the higher concentration exceeded the apparent Kms for SAM sulfation and glucuronidation but was less than the Kmfor hydroxylation. Simulation of SAM metabolism data was carried out with various enzyme distribution patterns and extended to include GAM metabolism. At both input concentrations, E washigh (0.94 at 134 μMand 0.7 at 295 μM) and unchanged during N and R, with SAM-sulfate (SAM-S) as the major metabolite and GAM-5G as the only detectable metabolite of GAM. Saturation of SAM sulfation occurred at the higher input SAM concentration as shown by a decrease in Eand a proportionally less increase in sulfation rates and proportionally more than expected increases in SAM hydroxylation and glucuronidation rates. At both SAM concentrations, the steady-state ratio of metabolite formation rates for SAM-S/SAM-G decreased when flow direction changed from N to R. An insignificant decrease in SAM-S/SAM-OH was observed at the low input SAM concentration, due to the small amount of SAM-OH formed and hence large variation in the ratio among the preparations, whereas at the high input SAM concentration, the decrease in SAM-S/SAM-OH with a change in flow direction from N to R was evident. The metabolite formation ratio, SAM-G/SAM-OH, however, was unchanged at both input concentrations and flow directions. The observed data suggest an anterior SAM sulfation system in relation to the glucuronidation and hydroxylation systems, which are distributed similarly. When the observations were compared to predictions from the enzyme-distributed models, the best prediction on SAM metabolism was given by a model which described sulfation activities anteriorly, glucuronidation activities evenly, and hydroxylation activities posteriorly (perivenous). When the model was used to predict data for SAM and GAM metabolism in once-through perfused rat livers at different input SAM concentrations, in the absence or presence of the sulfation inhibitor, 2,6-dichloro-4-nitrophenol (DCNP), the predictions were in close agreement with previously observed SAM data but failed to predict the exclusive formation of GAM-SG; rather, GAM-2S and GAM-5S were predicted as major sequential metabolites of SAM. The poor correlation for GAM metabolic data may be explained on the basis of subcellular enzyme localizations: the cytochromes P-450 and UDP-glucuronyltransferases, being membrane-bound enzymes, are more coupled for GAM formation and glucuronidation, when GAM was generated intracellularly. The present study suggests that subcompartmentalization of enzymes may need to be considered in hepatic modeling for better prediction of metabolic events.

An understanding of the role of enzyme localization of the liver on metabolite kinetics: A computer simulation

The metabolic sequence of drug, D,to its primary (MI)and terminal (MII)metabolites as mediated by enzymes Aand B,respectively, was chosen to illustrate metabolizing activities among hepatocytes in different regions of the liver lobule. Six models of distributions of the hepatocellular activities (intrinsic clearances for A and B) were defined with respect to the flow path in liver, and the concentrations D, MI,and MIIin the liver were simulated. The extent of sequential metabolism of the primary metabolite was compared for these six models of enzymic distributions. It was found that when the average hepatic intrinsic clearances of Aand Bwere high (almost complete extraction of both drug and primary metabolite during their single passage through the liver), the distributions of Aand Bwere not important determinants of metabolite kinetics. By contrast, when the average hepatic intrinsic clearances of A and Bwere both low,the distributions of Aand Bexerted profound effects on metabolite kinetics. The sensitivity to enzymic distribution in this region, however, was difficult to assess due to difficulties in detecting low levels of MIand MIIThe effects of enzymic distributions on metabolite disposition would be better detected in compounds (drug and metabolite) with intermediate extraction ratios.

Transport Is Not Rate-Limiting in Morphine Glucuronidation in the Single-Pass Perfused Rat Liver Preparation

Binding, transport, and metabolism are factors that influence morphine (M) removal in the rat liver. For M and the morphine 3β-glucuronide metabolite (M3G), modest binding existed with 4% bovine serum albumin (unbound fractions of 0.89 ± 0.07 and 0.98 ± 0.09, respectively), and there was partitioning of M into red blood cells. Transport studies of M (<750 μM) showed similar, concentration-independent uptake clearances (CLs) of 1.5 ml min-1 g-1 among zonal and homogeneous, isolated rat hepatocytes. Transport of M3G, ascertained in multiple indicator dilution studies at various steady-state M3G concentrations (10-262 μM), uncovered a low and concentration-independent influx clearance (<10% of flow rate). The outflow dilution curve of [3H]M3G was superimposable onto that of [14C]sucrose, the extracellular reference, displaying similarity in transit times (23.5 and 22.2 s), negligible biliary excretion, and almost complete dose recovery from perfusate. In contrast, M3G occurred abundantly in both perfusate and bile in single-pass perfusion studies of the precursor, M, and revealed a biliary clearance of formed M3G that was 12.3-fold that of preformed M3G, suggesting a sinusoidal, diffusional barrier for M3G. With increasing concentrations of M (9-474 μM), clearance decreased, and metabolism and biliary excretion displayed concentration-dependent kinetics. Fitting of the data to a physiologically based liver model revealed that M removal mechanisms were saturable, with a Km,met of 52.2 μM and Vmax,met of 58.8 nmol min-1 g-1 for metabolism, and a Km,ex of 41.2 μM and Vmax,ex of 8.1 nmol min-1 g-1 for excretion. Sinusoidal transport was not rate-limiting for M removal.

An enzyme-distributed system for lidocaine metabolism in the perfused rat liver preparation

The influence of enzymic distribution on lidocaine metabolism was investigated in the once-through perfused rat liver preparation. Low input concentrations of14C-lidocaine (1–2 μM) and preformed monoethylglycine xylidide (MEGX; 2.3–2.8 μM) were delivered by normal and retrograde flow directions to the liver preparations at 10 ml/min per liver. Upon reversal of normal to retrograde delivery of lidocaine, the rates at which lidocaine, MEGX, and glycine xylidide (GX) left the liver almost doubled, whereas the rates of appearance of (total) hydroxylated lidocaine and MEGX in bile and perfusate increased to lesser extents. Upon reversal of normal to retrograde delivery of preformed MEGX, the rates of appearance of MEGX and GX were virtually unchanged. Computer simulations on lidocaine and preformed MEGX metabolism were performed on both evenly distributed (“parallel tube” model) and enzyme-distributed systems. An even or parallel distribution of N-deethylation and hydroxylation activities for lidocaine metabolism failed to predict the observed increased hepatic availability of lidocaine. Rather, the distribution of a low-affinity, high-capacity N-deethylation system anterior to a high-affinity, lowcapacity hydroxylation system for lidocaine metabolism adequately predicted the increased hepatic availability of lidocaine. Further extension of these consistent enzyme-distributed models on the metabolism of lidocaine metabolites suggests that the N-deethylation and hydroxylation activities for the metabolism of lidocaine, MEGX, 3-hydroxyidocaine, and 3-hydroxy MEGX are not identically distributed. When these enzymedistributed models were appraised with reference to the “parallel tube” and “wellstirred” models of hepatic drug clearance, predictions from these.enzymedistributed models proved to be superior to the “parallel tube” and “well-stirred” models for the present data on lidocaine metabolites with normal and retrograde perfusions. Previously published data on lidocaine and MEGX metabolism after inputting 4 μg/ml (17 μM) lidocaine at flow rates of 10, 12, 14, and 16 ml/min were reexamined with respect to the adequacy of these enzyme-distributed models. They were found to be superior to the evenly-distributed or “parallel tube” model in predicting hepatic availability of lidocaine and the rate of appearance of MEGX. However, the enzyme-distributed systems were not as consistent as the “well-stirred” model in predicting lidocaine hepatic availability in these flow experiments.

Determination of diazepam and its metabolites by high-performance liquid chromatography and thin-layer chromatography

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4-hydroxy-N-desmethyldiazepam and 4-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.

A Comparative Investigation of Hepatic Clearance Models: Predictions of Metabolite Formation and Elimination

Liver clearance models serve to improve our understanding of the relationships between the physiological determinants and hepatic clearance and predict changes in the disposition of substrates when homeostasis of the organ is perturbed. Their ability to describe metabolism was presently extended to the sequential formation and elimination of primary (M1), secondary (M2), and tertiary (M3) metabolites during a single passage of drug (P) across the liver, under steady state and first-order conditions. The well-stirred model is distinct from other models in that metabolite formation and elimination is independent of enzymic distributions, the number of steps involved in metabolite formation, and the intrinsic clearances of the precursors. This model predicts that the extraction ratio of a formed primary metabolite derived from drug (E{M1, P}) is identical to that for the preformed primary metabolite (E{M1}), and that the extraction ratios of a secondary metabolite derived from drug (E{M2, P}) and primary metabolite (E{M2, M1}) or preformed secondary metabolite (E{M2}) are identical. For the more physiologically acceptable, parallel-tube and dispersion models, metabolite sequential elimination is highly influenced by the intrinsic clearances of the precursors and the enzymic distributions that mediate removal of precursor species and the metabolites. Furthermore, the extent of sequential metabolism recedes as the number of steps involved for metabolite formation increases. These models predict that E{M1, P}<E{M1}, and E{M2, P}<E{M2, M1}<E{M2}, with the magnitude of the changes being less for the dispersion model than for the parallel-tube model. Competing pathways that divert substrate from entering the sequential pathway were found to exert only minimal influence on the sequential pathway.

Competing pathways in drug metabolism. II. An identical, anterior enzymic distribution for 2- and 5-sulfoconjugation and a posterior localization for 5-glucuronidation of gentisamide in the rat liver

Gentisamide (2,5-dihydroxybenzamide, GAM), a substrate that forms two monosulfates at the 2 and 5 positions (GAM-2S and GAM-5S) and a monoglucuronide at the 5 position (GAM-5G), was delivered at 8 or 80 μM by normal (N) and retrograde (R) flows to the once-through rat liver preparation. At the lower (8 μM) input concentration, ratios of conjugate formation rate, GAM-5S/GAM-5G and GAM-2S/GAM-5G, were decreased significantly (4.01±1.42 to 2.93±0.99, and 1.13±0.65 to 0.66±0.41, respectively) whereas a small but significant increase in the steady-state extraction ratio, E (0.89±0.029 to 0.94±0.016), was observed upon changing the flow direction from N to R. At the higher input GAM concentration (80 μM), conjugate formation rate ratios were relatively constant for GAM-5S/GAM-5G (1.18±0.08 to 1.11±0.12) and GAM-2S/GAM-5G (0.33±0.05 to 0.31±0.05) upon changing flow direction from N to R, despite a slight increase in E from 0.87±0.023 to 0.91±0.016 was observed. These results suggest that the sulfotransferase activities responsible for 2- and 5-sulfoconjugations are identically distributed and localized anterior to 5-glucuronidation activities during a normal flow of substrate into the rat liver (entering the portal vein and exiting the hepatic vein), and that the presence of uneven distribution of conjugation activities is discriminated only at the lower input drug concentration. At high concentration (>Km for all systems), saturation of all pathways occurs, and other, anteriorly/identically distributed competing pathways would fail to perturb downstream intrahepatic drug concentrations arid the resultant conjugation rates. The lack of change in metabolic profiles renders the condition unsuitable for examination of uneven distribution of enzymes in the liver. These observations were generally predicted by theoretical enzymic models of consistent distribution patterns. Because 2- and 5-sulfation were mediated by systems of similar Km but different Vmax values, two possibilities, the same isozyme of sulfotransferase being involved in the formation of two enzyme-substrate complexes to form two distinctly different products or two isozymes of sulfotransferases of identical distribution, were discussed.

High-performance liquid chromatographic method for the direct determination of 4-methylumbelliferone and its glucuronide and sulfate conjugates: Application to studies in the single-pass in situ perfused rat intestine—liver preparation

A direct high-performance liquid chromatographic (HPLC) assay was developed for the separation and determination of 4-methylumbelliferone (4MU) and its glucuronide (MUG) and sulfate (MUS) conjugates in the cell-free perfusate (plasma) from in situ perfused rat intestine-liver preparation. In addition, a procedure was developed to extract and determine 4MU in the whole blood perfusate. Perfusate plasma containing an internal standard (umbelliferone) was precipitated with methanol (1:4, v/v), and injected into a reversed-phase HPLC system with gradient elution. 4MU and the same internal standard were also extracted directly from the whole blood perfusate with ethyl acetate and injected into a reversed-phase HPLC system with isocratic elution. Inter- and intra-day precision studies (n = 5 for each) for both the plasma and whole blood procedures demonstrated relative standard deviation of less than 10% at all concentrations studied. The compounds were stable in either the plasma or blood extracts at room temperature for up to 72 h. The procedures were successfully used to analyze perfusate samples obtained from the single-pass in situ perfusion of rat intestine-liver system with either trace (0.95 nM) or 32.3 microM concentrations of 4MU. The intestine was responsible for the formation of most of the MUG formed by the intestine-liver preparation during steady-state perfusion with either input concentration of 4MU.