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Sulfation and glucuronidation are two parallel pathways for the metabolism of phenolic substrates. Gentisamide (GAM) was used as a model compound to examine the effects of parallel competing pathways on drug disappearance and metabolite formation in the once-through perfused rat liver preparation. GAM was found to form one glucuronide (GAM-5G) and two sulfate (GAM-2S and GAM-5S) conjugates. These GAM conjugates were biosynthesized in recirculating rat liver preparations, and were isolated by preparative high-performance liquid chromatography. Specific incorporation of 35S-sodium sulfate and [14C]glucose into GAM sulfate and glucuronide conjugates revealed corresponding elution patterns as labeled GAM metabolites. Their identities were characterized by enzymatic and acid hydrolyses and by NMR spectroscopy. Gentisamide-5-sulfate (GAM-5S) and gentisamide-5-glucuronide (GAM-5G) are major metabolites, and gentisamide-2-sulfate (GAM-2S) is a minor metabolite. Single-pass rat liver perfusions were used to examine the effect of stepwise increases/decreases of input GAM concentration (CIn) on the extraction ratio (E) of GAM and formation of metabolites. The E of GAM remained constant (about 0.89) at input concentrations from 0.9 to 120 microM and decreased at CIn greater than 120 microM. Metabolite patterns, however, changed with GAM CIn, even when E was constant at CIn up to 120 microM. GAM-5S was present as the major metabolite of GAM at all GAM CInS in most liver preparations but the proportions of GAM-5S and GAM-2S decreased at increasing CIn; the proportion of GAM-5G, a minor metabolite at low CIn, increased with increasing CIn. Biliary excretion rates at steady state accounted for 5.3 +/- 2.7% (mean +/- S.D.) of the input rate: GAM-5G was the predominant metabolite found. Fitting the metabolic data (sum of the rates of efflux in bile and perfusate at steady state) and the logarithmic average drug concentration at the various CIn to the Michaelis-Menten equation furnished parameter estimates for the three metabolic pathways. The estimated Km and Vmax values were quite comparable: for GAM-2S formation, 22 microM and 287 nmol/min; for GAM-5S formation, 26 microM and 978 nmol/min; for GAM-5G formation, 71 microM and 1062 nmol/min, indicating that sulfation and glucuronidation are effective competing pathways of each other. In viewing the fate of GAM over the CIn range, a changing metabolic fate of GAM over a constant E was noted at CIn less than 120 microM: the sum of the rates of metabolite formation was proportional to CIn.(ABSTRACT TRUNCATED AT 400 WORDS)

alpha-Bromoisovalerylurea (BIU) is used as model substrate for studies on the pharmacokinetics of glutathione conjugation in vivo. Its metabolism in isolated rat hepatocytes is presently studied. A major part of the substrate was conjugated with glutathione, but also amidase-catalyzed hydrolysis occurred, resulting in the products urea and alpha-bromoisovaleric acid (BI). The amidase activity was located in the microsomal fraction of the rat liver. The product of hydrolysis, BI, also was conjugated efficiently with glutathione. In glutathione-depleted hepatocytes, no glutathione conjugates but only urea and BI were formed. A pronounced stereoselectivity in the metabolism of the BIU enantiomers was observed: (R)-BIU was conjugated with glutathione much faster than (S)-BIU. (S)-BIU was hydrolyzed substantially in the cells and the glutathione conjugate of the hydrolytic product, (S)-BI, could be detected. At high BIU concentrations (500 microM of the racemate) intracellular glutathione was seriously depleted; then, the cosubstrate availability most likely was the rate-limiting factor in the conjugation of BIU with glutathione. More urea was formed from (racemic) BIU in isolated rat hepatocytes in the present study than in the perfused liver and the intact rat in previous studies. This in vivo-in vitro difference is tentatively assigned to differences in glutathione availability in these systems. The results suggest that BI may also be a useful model substrate to study the kinetics of glutathione conjugation in vivo and in vitro.

4-Methylumbelliferyl sulfate (4MUS), a polar metabolite of 4-methylumbelliferone (4MU), is known to undergo desulfation and participate in futile cycling with 4MU. Unusual parabolic or increasing profiles of the steady-state extraction ratio (Ess) of 4MUS with respect to concentration in rat livers perfused with a red cell (20%)-albumin (1%) medium have been reported (Ratna et al., 1993). In order to study this unusual phenomenon, we examined the desulfation of 4MUS in the single-pass rat liver in the absence of albumin. We further employed a tubular-flow model to describe the present observations and data previously obtained on 4MUS in order to predict the effects of protein binding and enzymatic constants for conjugation/deconjugation on the hepatic processing of 4MUS and its metabolites. The net hepatic extraction ratio from albumin-free perfusate decreased from 0.465 to 0.326 when the 4MUS input concentration was increased from 122 to 908 microM; moreover, the unusual profiles previously observed for ESS with increasing concentration in albumin-containing perfusate were not apparent. The hepatic clearances and desulfation rates of 4MUS were essentially identical to those observed in the presence of albumin, when the latter were expressed in terms of unbound concentrations (unbound input and logarithmic average unbound concentration of the input and output blood). Initial modeling indicated that first nonlinear protein binding (dissociation constant KD of 93 microM) and then saturable desulfation (Km of 382 microM) were responsible for the unusual increasing and then decreasing trend of ESS with concentration in the presence of albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Perfusion of substrate via only the hepatic artery with simultaneous substrate-free perfusion of portal vein or hepatic vein [hepatic artery portal vein-hepatic artery hepatic vein] was used to examine the enzymic distribution of carboxylesterases towards the hydrolysis of enalapril to enalaprilat in the perfused rat liver preparation. In this single-pass method, [14C]enalapril was delivered by the hepatic artery at 2 ml/min into the liver, whereas drug-free perfusate entered the portal vein or hepatic vein at 10 ml/min for HAPV and HAHV perfusions, respectively. During steady state, a multiple indicator dose of 51Cr-labeled red blood cells (vascular marker), [58Co]EDTA (interstitial space marker, which behaves similarly to labeled tracer sucrose), and 3H2O was given into the hepatic artery. Labeled enalapril and the reference indicators entering via the hepatic artery will reach virtually all sinusoidal spaces during HAPV, and will be confined to the peripheral region during HAHV. By defining the steady-state extraction ratios of enalapril (Etot) and segregating the components of biliary excretion and metabolism, and by assessing the intracellular water spaces and the corresponding transit times during HPAV and HAHV, the metabolic sequestration rate constant (metabolic intrinsic clearance per unit accessible cell water space) for the periportal region/whole liver (HAHV/HAPV) was 0.344. The data suggest that the carboxylesterase activity for enalapril conversion to enalaprilat is primarily localized in the perihepatic venous region of the rat liver.

An isolated recirculating or single pass red cell-perfused rat kidney preparation (IPK) was used to examine the differential handling of renal metabolites. In single pass experiments, enalapril was primarily metabolized to its polar, dicarboxylic acid metabolite, enalaprilat, and its fractional excretion (FE) was less than unity, suggesting net reabsorption. Its steady-state extraction ratio decreased from 0.3 to 0.2 at concentrations of 1.06 to 12.7 microM, due to a saturation of enzymes for esterolysis. Enalaprilat administered to the IPK was excreted into urine in a concentration-independent (0.41-35.3 microM) fashion, with FE values approximating unity, suggesting net filtration. Differences in handling were observed for enalaprilat, as a metabolite formed from enalapril and as an administered (preformed) species in the single pass IPK, when tracer concentrations of [14C]enalapril and [3H]enalaprilat were given simultaneously. A comparison made between steady-state extraction ratio Ess[mi] [generated metabolite]/glomerular filtration rate (GFR) and Ess[pmi] [preformed metabolite]/GFR, respectively, revealed a 2-fold difference. The finding suggests the presence of a barrier for entry of enalaprilat into the kidney. Or else, in absence of the barrier, the opposite would be observed, that is, Ess [pmi]/GFR greater than Ess [mi]/GFR because preformed enalaprilat, in contrast to generated enalaprilat, undergoes filtration and utilizes facilitative transport carriers at the basolateral membrane. In recirculating IPKs which received simultaneously a tracer bolus dose of [14C]enalapril and [3H]enalaprilat, the FE values for generated [14C]enalaprilat were high and variable, decreasing with perfusion time and exceeding those for preformed [3H]enalaprilat, which approached unity with perfusion time. The variable FE values for [14C]enalaprilat are due to time-dependent contributions of circulating enalaprilat (which behaves identically to preformed enalaprilat) and the intrarenally generated enalaprilat. Hence, with renal drug metabolism, the conventional method of estimating urinary clearance (or Fe[mi]) for the metabolite [(total) excretion rate/midpoint plasma FE[mi] metabolite concentration] results in a greater metabolite clearance than that predicted from the administration of preformed metabolite.

Both [14C]phenacetin and [3H]acetaminophen in tracer concentrations were perfused simultaneously once through the rat liver preparation at a constant perfusate flow rate (10 ml/min), and the rates of appearance of [14C]acetaminophen and [3H]acetaminophen in the effluent were compared. The data indicated that the extraction ratio of [14C]acetaminophen derived from [14C]phenacetin was smaller than that of the preformed [3H]acetaminophen added to the input perfusate (exogenously), i.e., the availability of the metabolite formed in situ was higher than the availability obtained when the metabolite was presented in the input blood. The observed availability of the acetaminophen derived from phenacetin was usually greater than that predicted by a "well-stirred" model and less than that predicted by a "parallel tube" model of hepatic drug clearance; the former model describes the liver as a well-stirred compartment with the drug in liver in equilibrium with that in the hepatic venous blood, and the latter model describes the liver as a group of identical and parallel uints with enzymes distributed evenly in hepatocytes lining the tubes. We conclude that the liver may be viewed as an imperfectly mixed compartment with regard to the availability of the metabolite which is generated from a precursor.

The role of zonal distributions of metabolic activities (sulfation and glucuronidation) in the liver on the kinetics of harmol conjugation was investigated. A computer simulation approach was adopted to better understand the effects of distributions of these conjugation activities in competition for a common drug substrate. Several distributions of the sulfation and glucuronidation systems were defined with respect to the flow path of blood; the conjugation activities along the flow path were in turn translated as time elapsed after entry of the substrate via blood into the organ. Realistic values of Km and Vmax for the sulfation and glucuronidation systems were assigned in the simulations. Directional flow, namely, normal vs. retrograde delivery of substrate was used as an additional variable. When the "center" of distribution of the sulfation system was anterior to that for glucuronidation, the steady-state hepatic extraction ratio of harmol (E) would increase, whereas the ratio of the steady-state rates of formation of harmol sulfate to harmol glucuronide (S/G ratio) would decrease when harmol was presented in a reversed direction (via retrograde perfusion), as compared with normal directional flow to the liver. The converse was true for an anterior distribution of the glucuronidation system; E would decrease, whereas S/G would increase with retrograde perfusion. To evaluate such zonal differences experimentally, perfusion studies were conducted in livers of male Wistar rats. A constant concentration of harmol (50 microM) was delivered under constant hepatic flow rate (10 ml/min/liver) by normal and retrograde perfusions to the same rat liver preparation. The steady-state hepatic E was higher (P less than .005) during retrograde perfusion than during normal perfusion, whereas the S/G ratio was significantly decreased (P less than .0005). The observations suggest an intercellular difference in the distribution of the two conjugating systems and are consistent with the view of the center of distribution for the sulfation system being anterior to the center of distribution for the glucuronidation system along the normal flow path of blood in the liver.

Glycine conjugation activity toward benzoic acid (BA) was studied in the single-pass perfused rat liver preparation. The steady-state hepatic extraction ratio for the portal vein (PV) perfused liver (at 10 ml/min) was maximal (0.6) at input concentrations < 40 microM among perfusions varying from tracer to 700 microM. Glycine conjugation was the predominant pathway; the kinetic parameters estimated after appropriately correcting for plasma protein binding (KA = 8.37 x 10(3) M-1 and 1.9 sites) revealed a low Km (12 microM) and a moderately high Vmax (101 nmol.min-1.g-1 liver) system. Biliary excretion of BA and its glycine-conjugated metabolite, hippuric acid, was minimal. Under first-order conditions (input concentration < 2 microM), the method of HAPV and HAHV perfusion (trace [14C]benzoate delivered via the hepatic artery (HA) at 2 ml/min and blank perfusate via the portal vein (PV) or hepatic vein (HV) at 10 ml/min) was used to examine the localization of glycine conjugation activity toward BA. During steady state, a multiple indicator dose of 51Cr-labeled red blood cells (vascular marker), [58Co]EDTA (interstitial space marker, which behaves similar to labeled tracer sucrose) and 3H2O (cellular marker) was injected as a bolus into the HA. Values of the extraction ratio of BA for HAPV perfusion (0.59 +/- 0.09) were dramatically reduced during HAHV perfusion (0.061 +/- 0.033, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetics of influx, efflux and removal of salicylamide under steady-state conditions (input concentration, 9-870 microM) were studied with the single pass erythrocyte-perfused rat liver in the absence of albumin (12 ml min-1). A substantial distribution of salicylamide into red blood cells (red cell/plasma ratio, 3.5) was observed. During steady state, a bolus dose containing multiple indicators (51Cr-labeled red cells (vascular space marker), [3H]sucrose (interstitial space marker), D2O (cellular space marker) and added [14C]salicylamide tracer) was injected into the portal vein. The steady-state hepatic salicylamide extraction ratio decreased from 0.99 to 0.4 over the concentration range used. The extraction ratio for bulk salicylamide was similar to 1 minus the integral of the fractional outflow recovery of unchanged tracer [14C]salicylamide (or [1-F] where F is the availability). Modeling of the indicator dilution outflow data revealed an extremely rapid (flow-limited) influx and efflux for salicylamide that was independent of the partitioning of salicylamide into red blood cells. The decrease in extraction ratio was due solely to saturation of the metabolic processes, shown previously to be sulfation, glucuronidation and hydroxylation. The sequestration rate constant, representing the pooled constant for all of the metabolic pathways, decreased from 0.45 to 0.035 sec-1 with increase in concentration. From the spectrum of its change with concentration, calculated values for a corresponding apparent Vmax and Km were 17.5 nmol sec-1 ml-1 cellular water and 27 microM, respectively. The cellular distribution space for salicylamide was exceedingly large, 20 times the size of the cellular water space, at trace levels, and fell to a constant level (5 times the cellular water space) with increasing concentration. The phenomenon, first observed for n-propanol (Goresky et al., Am. J Physiol. 244: G215-G244, 1983a), is explained by a nonsaturable (partition coefficient, 3.8-5) and a saturable binding (binding site concentration, 352-98 microM) component in the tissue. The binding effects inverted the order of elution of the parent and metabolite profiles; the outflow emergence of metabolites began earlier than that of the parent compound, salicylamide.

Although normal-retrograde liver perfusion allows definition of relative distribution patterns of multienzyme systems involved in parallel, competing and sequential metabolic pathways, the technique fails to describe localization patterns for unienzyme systems. A novel method of perfusion, which can describe the relative zonal enrichment of drug metabolizing activity of unienzyme systems in periportal region and in the entire liver, is described. The rat liver was perfused once-through with combined hepatic arterial (2 ml/min/liver-containing drug) and portal or hepatic venous (10 ml/min/liver, without drug) flows [HAPV and HAHV]. Because of known enrichment of phenacetin O-deethylation and acetaminophen sulfation activities in the perihepatic venous and periportal regions, respectively, metabolic data derived with these compounds were used for validation of the method. The overall steady-state hepatic extraction ratio of [3H]phenacetin during HAPV (0.88-0.98) was decreased markedly during HAHV (0.06-0.47), but was reduced to a lesser extent for preformed [3H]acetaminophen (0.57-0.86 for HAPV to 0.23-0.62 for HAHV). Because a comparison of metabolic activities encountered by substrate(s) [estimated from extraction ratios] required normalization to the intracellular water spaces accessed during both HAPV and HAHV, the multiple indicator dilution technique (a bolus injection of noneliminated reference materials: 51Cr-labeled RBC, 125I-labeled albumin, [14C]sucrose and 3H2O) into the hepatic artery during both HAPV and HAHV was used, with outflow profiles from the hepatic vein or portal vein characterized by rapid serial collections. Sinusoidal blood volume, albumin accessible Disse space, sucrose accessible Disse space and the estimated intracellular water space reached during HAHV were 48, 56, 53 and 33% those during HAPV. The ratios of intrinsic clearances per volume of cell water space for HAHV/HAPV, representing periportal activity/whole liver activity were: phenacetin O-deethylation, 0.34 and acetaminophen sulfation, 1.13. These findings validate the use of HAHV and HAPV perfusion for the examination of relative abundances of enzymic activities across the liver acinus for substrates metabolized by unienzyme systems.

The elimination of [14C]acetaminophen which was formed from [14C]phenacetin was slower than that for the preformed metabolite, [3H]acetaminophen. The observation had been attributed to the uneven distribution of enzyme systems for O-deethylation in the formation of acetaminophen and sulfate-conjugation of acetaminophen in the liver parenchyma. In this study, retrograde perfusion which reversed not only the direction of hepatic flow into the liver but also the location of enzyme systems with respect to the flow path, was used to examine the elimination kinetics of [14C]acetaminophen and [3H]acetaminophen. In the same rat liver preparation, both [14C[phenacetin and [3H]acetaminophen in tracer concentrations were delivered simultaneously into the perfused rat liver in situ preparation with normal directional flow (into the portal vein and out of the hepatic vein). The direction of flow was then reversed to retrograde perfusion (into the hepatic vein and out of the portal vein) which was later reverted back to normal perfusion. The elimination of [14C]acetaminophen was again slower than the elimination of [3H]acetaminophen during normal perfusion, but the elimination kinetics were virtually identical for both metabolite species during retrograde perfusion. This finding confirmed our previous hypothesis that sulfate-conjugation occurred predominantly in the periportal region while O-deethylation occurred preferentially in the centrilobular region of the liver and showed that retrograde perfusion was a useful probe in the investigation of the uneven distribution of hepatic drug metabolizing enzyme systems.

The role of hepatic intrinsic clearance for metabolite formation from various precursors on subsequent metabolite elimination was was investigated in the once-through perfused rat liver preparation. Two pairs of acetaminophen precursors: [14C] phenacetin-d5 and [3H] phenacetin-do, [14C] acetanilide and [3H] phenacetin were delivered by constant flow (10 ml/min/liver) either by normal or retrograde perfusion to the rat liver preparations. The extents of acetaminophen sulfation were compared within the same preparation. The data showed that the higher the hepatocellular activity (intrinsic clearance) for acetaminophen formation, the greater the extent of subsequent acetaminophen sulfation. The findings were explained on the basis of blood transit time and metabolite "duration time." Because of blood having only a finite transit time in liver, the longer the drug requires for metabolite formation, the less time will remain for metabolite sulfation and the less will be the degree of subsequent sulfation. Conversely, when the drug forms the primary metabolite rapidly, a longer time will remain for the metabolite to be sulfated in liver to result in a greater degree of metabolite sulfation. Finally, the effects of hepatic intrinsic clearances for metabolite formation and zonal distribution of enzyme systems for metabolite formation and elimination in liver are discussed.

A new procedure, namely the in situ perfused rat intestine-liver preparation, was introduced to examine the roles of the intestine and the liver in the elimination of enalapril, a new angiotensin-converting enzyme inhibitor. The in situ perfused rat intestine preparation was used to determine the rate and extent of enalapril absorption after an-intraduodenal dose. In the former technique, enalapril in blood perfusate (10 ml/min) was delivered via the superior mesenteric artery into the once-through perfused rat intestine-liver preparation, with sampling effected in reservoir, portal vein and hepatic vein. The ease of sampling, proximal and distal to the intestine and liver, allowed the direct estimation of the extraction ratios by the intestine and the liver. The steady-state intestinal extraction ratio of enalapril was small (0.04 +/- 0.066) compared to that for the liver (0.74 +/- 0.06), indicating that the liver was responsible for most of the hydrolytic conversion of enalapril to its pharmacologically active diacid metabolite, enalaprilat. Moreover, no trend in the values of the extraction ratios by both organs was apparent among the input concentrations of enalapril (0.55, 2.6 and 13.3 microM) used. Portal venous plasma consisted mainly of enalapril and was devoid of enalaprilat, whereas both enalapril and enalaprilat were detected in bile and hepatic venous plasma. With the latter technique, an intraduodenal injection of a tracer dose of [14C]enalapril (0.14-0.39 mumol) was made close to the pyloric sphinctor, whereas the intestine preparation was recirculated (7.5 ml/min) with blank perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies in the once-through perfused rat liver preparation have shown that the techniques of normal and retrograde delivery of substrate and computer simulation of enzyme distributions along the sinusoidal flow path in liver were useful in delineating the relative distributions of sulfation and glucuronidation activities for harmol metabolism (Pang et al., J. Pharmacol. Exp. Ther. 224: 647-653, 1983). The observed steady-state hepatic extraction ratios of harmol and the steady-state formation rates of harmol sulfate and harmol glucuronide were consistent with three enzyme-distribution models which described a proximal localization of sulfation activity and a more distal distribution of glucuronidation activities. The present study was a further refinement on the definition of the distribution of these activities. The experimental approach used included the perturbation of delivery of harmol (10 microM) at 8, 12 and 16 ml/min by normal flow, and the alternation of normal and retrograde directional flows for delivery of harmol (10 microM) at constant hepatic blood flows (8, 12 or 16 ml/min) in the single-pass perfused rat liver preparation. The observed steady-state sulfation and glucuronidation rates were compared against predicted values afforded by models previously shown to be adequate and additional enzyme-distributed models. The observed and predicted data point to a periportal distribution of sulfation activity and an even distribution of glucuronidation activity for the metabolism of harmol.

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of [(R)- and (S)-]2-bromoisovalerylurea (BIU), yielding bromoisovaleric acid (BI) and urea, have been observed in the rat in vivo and in isolated rat hepatocytes. The metabolism of enantiomeric (R)- and (S)-BIU was presently examined in the single-pass perfused rat liver with varying input concentrations (8-250 microM). Steady-state hepatic extraction ratios for (R)-BIU (0.6) were constant and higher than those for (S)-BIU, whose extraction ratio decreased from 0.36 (8 microM) to 0.23 (236 microM). (R)- and (S)-BIU were excreted unchanged only minimally into bile. [14C-Urea](R)-BIU underwent amidase-catalyzed hydrolysis to yield [14C]urea (15-24% of rate in) and conjugation to form the (S)-glutathionyl conjugate (31-35% of rate in); two metabolites, most likely the cysteinyl and dipeptide conjugates of BIU (10% of rate in), were found. [3H-Isovaleryl](S)-BIU formed much less amidase-hydrolyzed product, [3H]BI (1-2% of rate in) less (R)-glutathionyl conjugate (9-18% of rate in), but appreciable amounts (14-17% of rate in) of three other metabolites, of which two were most likely the cysteinyl and glycinylcysteinyl conjugates of BIU. When the glutathione conjugation products (glutathione, cysteine and cysteinylglycine conjugates) were summed, the total glutathione conjugation rate for (R)-BIU (44% of rate in) exceeded that for (S)-BIU (34 to 24% of rate in). Fitting of data to the Michaelis-Menten equation revealed similar Km for glutathione conjugation, but a 2-fold higher Vmax for (R)-BIU.(ABSTRACT TRUNCATED AT 250 WORDS)

In murine liver, temazepam (TZ) and nordiazepam (NZ) are mainly metabolized via N-demethylation and C3-hydroxylation, respectively, to form a common metabolite, oxazepam (OZ), which is then glucuronidated. With these precursors, we tested the hypotheses that the sequential metabolism of a primary metabolite (OZ) is less than that of the preformed metabolite and is dependent on the effective intrinsic clearance (unbound fraction x intrinsic clearance) of its precursor, as predicted by the parallel tube and dispersion models of hepatic drug clearances. Mouse livers were perfused with tracer concentrations of [14C]-NZ, [14C]-TZ and [3H]NZ in a single-pass fashion (2.5 ml/min). The steady-state extraction ratio (E) of [3H]NZ, [14C]NZ and [14C]TZ were 0.29, 0.40 and 0.49, respectively (P < .01), whereas the fractional metabolism (formation rate/total elimination rate of drug) of [3H]-NZ, [14C]NZ and [14C]TZ to form OZ was 0.39, 0.79 and 0.68, respectively. Values of E of [3H]NZ and [14C]NZ and fractional metabolism for OZ formation had differed because of a kinetic isotope effect (around 3.5) that affected the C3-hydroxylation of [3H]NZ. The extraction ratios of OZ (E[OZ,P]) arising from [14C]-NZ and [14C]TZ were both 0.056, and were less than that for preformed OZ (E[OZ]), previously found to be 0.125. The parameter E[OZ,P] was poorly correlated with the extraction ratio of the precursor, was overestimated by the parallel tube and dispersion models, but was highly correlated with the effective intrinsic clearance of the precursor (unbound fraction x intrinsic clearance).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetic theory dictates that the extent of ensuing metabolism of a formed metabolite during drug transit through the liver is influenced by the number of consecutive reactions required for its genesis and the total intrinsic clearances of the precursors. This hypothesis was tested in the perfused murine liver by examining the successive conversion of the precursor diazepam (DZ) to its primary metabolite nordiazepam (NZ), and then the secondary metabolite oxazepam (OZ) and, finally, the tertiary metabolite, the oxazepam glucuronides. The concomitant C3-hydroxylation of DZ to temazepam, which can also be N-demethylated to form OZ, was minimal. The hepatic extraction ratios of NZ (E[NZ,DZ]) and OZ (E[OX,DZ]) after administration of [14C]DZ were compared to those obtained previously from [14C]NZ (E[NZ] and E[OZ,NZ]) and [3H]OZ (E[OZ]). The ability of three hepatic clearance models, the well-stirred, parallel-tube and dispersion models, to predict the experimental E[NZ,DZ] and E[OZ,DZ] was evaluated. DZ was highly extracted by the murine liver (E[DZ] = 0.95). The metabolism of NZ, generated in situ from DZ, was greater than that of preformed NZ (E[NZ,DZ] = 0.51; E[NZ] = 0.4), whereas E[OZ,DZ] (0.066) was similar to E[OZ,DZ] (0.056) and less than E[OZ] (0.0125). The unexpected observation of E[NZ,DZ] > E[NZ] may be explained by the coupling of N-demethylation and C3-hydroxylation/glucuronidation reactions or by a sequestration of hydrophobic substrates within the enzymic space, favoring sequential metabolism of products formed in situ. The atypical kinetic behavior of generated NZ may have also influenced the ensuing metabolic fate of its product, OZ, such that E[OZ,NZ] approximately E[OZ,DZ].(ABSTRACT TRUNCATED AT 250 WORDS)

Previous rat liver perfusion studies had shown a preferential derecruitment of downstream (phenacetin O-de-ethylation) in relation to upstream (acetaminophen sulfation) hepatocyte activities with reductions in prograde (normal) flow, with substrates entering from the portal vein and exiting the hepatic vein. The reduction in metabolic activities was found associated with a reduction in accessible intracellular water space (Pang et al., J. Pharmacokinet. Biopharm. 16: 595, 1988). In the present studies, we investigated the changes in metabolic activities of the liver and its accessible spaces during retrograde flow (substrates entering from the hepatic vein and exiting the portal vein), an approach used to probe zonal heterogeneity in the liver. The multiple indicator dilution technique was used to gain information concerning the accessible sinusoidal blood volume, Disse space, and the intracellular water space; once-through perfusion with tracer [3H]acetaminophen and [14C]phenacetin was performed simultaneously within the same liver, to estimate metabolic activities at the periportal and perihepatic venous regions of the liver, respectively, at the designated flows. The estimated sinusoidal blood volume, total albumin and sucrose distribution spaces, the Disse space and the accessible albumin and sucrose Disse spaces showed weak but positive correlations with retrograde perfusate flow rate. The values were substantially greater than those for rat livers perfused with normal (prograde) flows, suggesting an increase in the vascular space and accessible Disse space of the liver during retrograde flow. The total water and accessible intracellular water spaces also showed weak but positive correlations with retrograde perfusate flow, and the average intracellular water space (which is also accessed by the substrates) at both retrograde flows of 8 and 12 ml/min was 0.52 +/- 0.08 and 0.55 +/- 0.08 ml/g liver, respectively, values that were not statistically different. When the intracellular water space was plotted vs. water flow rate (ml water/g liver), a weakly positive correlation was again found, suggesting that over the flow range, the accessible intracellular water space decreased in a very gradual and linear fashion with a reduction in water flow rate. When the estimated intracellular water space for retrograde flow was compared with values obtained for the same flow rate in a prograde fashion, slightly lower values were obtained. After consideration of the effect of the distended vascular and interstitial spaces on the weights of the liver, the values obtained for retrograde and prograde flow rates were closely similar. In both cases, it appeared that with reduction in flow, the accessible intracellular water spaces were reduced, regardless of the flow direction.(ABSTRACT TRUNCATED AT 400 WORDS)

Gentisamide (GAM), the hydroxylated metabolite of salicylamide (SAM), underwent sequential metabolism, and GAM-5-glucuronide was the only metabolite detected in the single-pass in situ rat liver preparation (10 ml/min/liver) perfused at varying SAM steady-state input concentrations (CIn). The exclusive formation of GAM-5-glucuronide was unexpected, because GAM, when administered to the rat liver, formed predominantly monosulfate conjugates at the 5- and 2-positions (Morris et al., J. Pharmacol. Exp. Ther. 245: 614-624, 1988). Kinetic parameters obtained from single-pass studies for SAM sulfation (CIn increasing from 31 to 347 microM) and glucuronidation and hydroxylation (CIn decreasing from 1383 to 130 microM, with 0.85 mM SO4(2-), and CIn increasing from 32 to 800 microM in an absence of SO4(2-), with 2,6-dichloro-4-nitrophenol, a sulfation inhibitor) revealed sulfation as a high-affinity, high-capacity pathway, glucuronidation as a lower-affinity, high-capacity pathway and hydroxylation as a low-affinity, low-capacity pathway. However, these parameters would not explain the exclusive glucuronidation of GAM when generated from SAM. Rather the zonal localization of metabolizing activities [a periportal sulfation, evenly distributed glucuronidation, and perivenous hydroxylation system (Xu and Pang, J. Pharmacokinet. Biopharm. 17: 645-671, 1989; Morris et al., J. Pharmacokinet. Biopharm. 16: 633-656, 1988)] could explain the complete lack of sulfation in SAM sequential metabolism. The different metabolite patterns arising from the administrations of a metabolite precursor (SAM) versus a preformed metabolite (GAM), due to the proximity of enzymes for formation and sequential metabolism, may serve to explain the differential toxic or pharmacologic effects observed in other precursor-metabolite pairs.