Acinar factors in drug processing: protein binding, futile cycling, and cosubstrate.
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Animals↗
Biomedical subjects
K S Pang
Publications and source records attributed to K S Pang.
Glutathione depletion kinetics with acetaminophen. A simulation study.
A tubular-flow model with known published parameters on glutathione (GSH) synthesis, degradation, and transport was developed to examine the distributed-in-space bimolecular reaction of GSH conjugation with acceptor substrates. Simulations were performed to obtain the vascular and intracellular GSH concentrations in the absence and presence of acetaminophen. Zonal localization of GSH was found to be effectively modified upon varying the activities for GSH synthesis and degradation along the sinusoidal flow path. A periportal (zone 1) GSH distribution resulted when GSH synthetic activity was distributed anterior to the degradation activity (models A and D); a perivenous (zone 3) GSH enrichment existed when these activities were reversed (model B), whereas when the synthetic and degradation activities for GSH were homogeneously distributed (model C), GSH concentration was unchanged in all zones. Although the zonation of GSH was model-dependent (models A-D ), only minor differences were found to exist for the length-averaged tissue GSH concentration (5.8-6 mumol/g liver) and the outflow of the liver (approximately 15 microM). With acetaminophen, a substrate known to deplete GSH via its reactive intermediate, N-acetyl-p-quinoneimine (NAPQI), acinar GSH patterns were not greatly perturbed at concentrations < 1 mM. At 10 mM acetaminophen, however, differential patterns of GSH zonal depletion were observed among models, although there was virtually no difference in the length-averaged intracellular GSH concentration (3 mumol/g liver) nor in the formation of the acetaminophen GSH adduct, with the latter being rate-limited by the bioactivation of acetaminophen to NAPQI.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetaminophen↗
Sulfation of acetaminophen by the perfused rat liver: the effect of red blood cell carriage.
Acetaminophen uptake and conversion in the perfused rat liver to acetaminophen sulfate was studied with the multiple indicator dilution technique (MID). Because acetaminophen is avidly bound to red blood cells and not albumin, a pre-equilibrated MID dose containing the noneliminated references (51Cr-labeled red blood cells [RBC, a vascular reference], [58Co]EDTA [a small molecular weight interstitial reference that does not enter cells], and D2O [a cellular reference]) and [3H]-acetaminophen was introduced into the portal vein of the single-pass perfused rat liver (1 mg/L acetaminophen) under varying conditions of hematocrit, with observation of timed outflow profiles in the hepatic venous blood. The [3H]acetaminophen curve exhibited an early high peak, paralleling that for red cells and varying with hematocrit, followed by a prolonged decline, with the late appearance of acetaminophen sulfate product; the early peak disappeared when red cells were absent from the dose and perfusate. Analysis demonstrated a slow release of acetaminophen from the red blood cells and rapid liver cell entry, so that red cell binding was displayed as a red cell carriage effect that reduced the rate of liver cell entry and hence of sulfation of [3H]-acetaminophen. The liver cells exhibited a concomitant very low permeability to product acetaminophen sulfate, leading to protracted product outflow curves. An inferred slow efflux-mediated storage phenomenon for product was found to evolve as a result.
Acetaminophen↗
Kinetics of sequential metabolism. Contribution of parallel, primary metabolic pathways to the formation of a common, secondary metabolite.
Interpretation of rate constants in the sequential metabolism of two different primary metabolites (MIA and MIB) for formation of a common, secondary metabolite (MII) after drug administration requires theoretical development of formulations that govern mass transfer during intravenous and oral administrations. Two cases (a and b) were presently considered for metabolism occurring only in the first-pass organs (intestine and liver) for flow-limited drugs and primary and secondary metabolites: (case a) wherein drug formed only the two primary metabolites, with the fractions of total body clearance that formed MIA and MIB, being denoted by f1 and f2, respectively, and (case b) wherein other additional elimination pathways for drug were present. MIA and MIB only partially formed MII (denoted by fMIA and fMIB, respectively), because provision was made for alternate elimination pathways; the fractional clearance in the formation of MII from MIA and MIB were fMIA and fMIB, respectively. Drug was metabolized to MIA than MII within the gut lumen with oral drug administration; the MIA and MII formed were further absorbed. Triangular matrices were found to result from mass transfer equations for first-order conditions with oral and intravenous administrations. Upon inversion of the matrices, the areas under the curve for drug and metabolite species were obtained after multiplication by the administered dose and division by the volume of the species considered. However, the dose-corrected area under the curve was used as the basis for comparison. Case-independent solutions were obtained for the fractions absorbed (Fa, FaMIA, FaMIB) and the availabilities (F, F(MIA), F(MIB)) of drug and the primary metabolites, and for f1, f2, f1/f2, fMIA/fMIB, and (f1fMIA)/(f2fMIB) (ratio of effective clearances of MII formation from D via MIA and MIB). Case-dependent solutions also existed. For case a (f1 + f2 = 1), the fraction of total body clearance that formed MIA (f1) or MIB (f2) was solved with the area under the curve of MII after intravenous D, MIA, and MIB administrations. For case b, however, the same constants were obtained after greater manipulation, and entailed oral administration of the metabolites. Although solutions for the ratios of f1/f2 and (f1fMIA)/(f2fMIB) were found, the fractional clearances in formation of MII from MIA (fMIA) and MIB (fMIB) were, however, not provided in both cases unless MII was completely absorbed.
Administration, Oral↗
Benzoic acid glycine conjugation in the isolated perfused rat kidney.
The fate of varying input concentrations (0.002-372 microM) of benzoic acid was examined in the single-pass isolated perfused rat kidney preparation under constant flow rate (8 ml min-1.organ-1). With an increasing concentration of benzoate, the steady-state renal extraction ratio decreased from 0.24 to 0.1. Little unchanged drug was found in the urine; the urinary clearance of benzoate was low (0.018 ml-1 min-1.g-1) and concentration-independent, yielding a rather constant fractional excretion of approximately 0.2. Metabolic clearance, due primarily to conjugation with glycine to form hippuric acid, constituted the majority of total renal clearance, and this decreased with concentration. These divergent trends for the metabolic and urinary clearance with concentration suggest that benzoate net influx across the basolateral membrane has not been saturated. Upon fitting of the hippurate formation rates vs. the plasma unbound logarithmic average concentrations of benzoate, overall kinetic constants (KM = 5.3 microM and Vmax = 195 nmol min-1.g-1) that likely reflect glycine conjugation were obtained. The formed hippurate either returned to the venous circulation or was excreted into urine without further biotransformation; the apparent renal extraction ratio (excretion rate/formation rate of hippurate) was quite high (approximately 0.48). Avid glycine conjugation and hippurate excretion thus occurred with administration of benzoic acid to the isolated perfused rat kidney.
Absorption↗
Transport, binding, and metabolism of sulfate conjugates in the liver.
Sulfate conjugates are a heterogeneous class of polar, anionic metabolites that result from the conjugation of endogenous and exogenous compounds. Sulfate conjugates exhibit a high degree of binding to albumin, the extent of which usually exceeds those of their parent compounds. Preponderant direct and indirect evidence suggests that sulfation activity is slightly higher in the periportal than in the perivenous (centrilobular) region of the liver, but recent immunohistochemical studies imply that specific isoforms of the sulfotransferases may also be preferentially localized in the perivenous region. Entry of sulfate conjugates into the liver cell is poor unless discrete carriers are present. Although known transport carriers exist for the sulfated bile acids, the specificity of the carriers for drug sulfate conjugates is presently unknown. The removal of sulfates is usually by way of biliary excretion while, on occasion, sulfates can be desulfated and participate in futile cycling with their parent compounds. The binding, transport, and hepatic elimination of various drug sulfate conjugates are examined. Non-recirculating studies carried out in the perfused rat liver with the multiple indicator dilution technique under varying input sulfate conjugate concentrations have provided essential information on the effects of vascular (red blood cells and plasma protein) binding on transport and removal of the conjugates. These studies clearly demonstrate the need to study protein binding, transmembrane transfer characteristics across the liver basolateral (sinusoidal) and canalicular membranes, and enzyme zonation in a distributed-in-space fashion in order to properly define the handling of sulfate conjugates in the liver.
Animals↗
Demonstration of rapid entry and a cellular binding space for salicylamide in perfused rat liver: a multiple indicator dilution study.
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.
Animals↗
Role of the hepatic artery in the metabolism of phenacetin and acetaminophen: intravital microscopic and multiple-indicator dilution study in perfused rat liver.
We studied the pattern of intermixing of the hepatic arterial and portal venous flows in a perfused rat liver preparation under constant flow (12 ml/min) with intravital epifluorescent microscopy; changes in the steady state extraction ratio of carbon 14-labeled phenacetin and tritiated acetaminophen, probes metabolized primarily in perivenous and periportal regions of the rat liver, respectively; and the spaces accessed by noneliminated reference indicators introduced as a bolus into the hepatic artery and portal vein at different hepatic arterial/portal venous flow regimens of 0:12, 2:10 and 4:8. The sinusoidal velocities for the hepatic arterial- and portal venous (hepatic arterial/portal venous flow at 4:8)-infused fluorescein isothiocyanate-erythrocytes (100 microliters/min) were 327 +/- 78 and 301 +/- 63 microns/sec, respectively, and the velocity for the solely portal venous-perfused liver (12 ml/min) was 347 +/- 74 microns/sec; the flow-weighted sinusoidal velocity was highly correlated to the sinusoidal volume for the dually perfused rat liver. Small but significant decreases in the extraction ratio of [14C]phenacetin (from 0.989 to 0.984 and 0.980) and tritiated acetaminophen (from 0.631 to 0.607 to 0.563), delivered simultaneously into the hepatic artery and portal vein, were observed with an increment of hepatic arterial flow within the same liver preparation; oxygen consumption rate also fell slightly, in parallel fashion. When a multiple-indicator dilution dose containing chromium 51-labeled RBCs, iodine 125-labeled albumin and tritiated water or [14C]urea was injected into the hepatic artery (which accesses both the peribiliary capillary plexus [nonsinusoidal] and the sinusoidal bed) and portal vein (which enters only the sinusoids) at 10-min intervals within each steady state, the blood volume, total albumin space, albumin Disse space, total water and parenchymal cellular water spaces were unchanged after portal venous injection for all hepatic arterial/portal venous flow ratios, suggesting that the arterial flow is ineffective in perturbing average sinusoidal flow dynamics. However, slightly larger total water spaces were obtained with hepatic arterial injection. This excess water space was almost completely accounted for by the "nonsinusoidal" extravascular space associated with the peribiliary capillary plexus; it averaged 0.03 ml/gm and was independent of flow. The anomaly, a reduced flow-weighted sinusoidal velocity for the dually perfused liver, an unchanged diameter of the terminal hepatic venule (32 microns) among the hepatic arterial/portal venous flow ratios and the reduction in the extraction ratio of the drug probes and oxygen consumption rates suggest that some of the arterial flow must have entered the sinusoids somewhat downstream.
Acetaminophen↗
Salicylamide sulfate cell entry in perfused rat liver: a multiple-indicator dilution study.
The hepatocellular entry of salicylamide sulfate conjugate, which binds to both red blood cells and albumin, was examined with the multiple-indicator dilution technique in the perfused rat liver, with medium containing both 20% red cells and 1% albumin (set A), red cells only (set B), albumin only (set C) and neither red cells nor albumin (set D). [14C]Salicylamide sulfate, 51Cr-labeled red cells (a vascular reference), 125I-labeled albumin, [3H]sucrose or [58Co] ethylenediaminetetraacetic acid (EDTA) (high and low molecular weight interstitial references, respectively) and 3H2O or D2O (a cellular reference) were injected as a bolus into the portal vein. Among all sets of outflow data, the earliest immediate vascular recoveries for the [14C] salicylamide sulfate were lower than those for the vascular reference. For sets A and B, the upslopes precessed those for the 58Co-EDTA curve, then crossed over the 58Co-EDTA curves, with lower magnitude peaks occurring at the same time as those for labeled albumin, whereas for sets C and D, in which red cells were absent, the upslopes of [14C]salicylamide sulfate lagged behind those for labeled albumin and [3H]sucrose, reaching lower magnitude peaks coincidental in time with those for labeled sucrose. The precession of the [14C]salicylamide sulfate curve over 58Co-EDTA or [3H]sucrose in the presence (sets A and B) but not in the absence (sets C and D) of red cells and the absence of precession with albumin alone (set C) suggest that a red cell effect on the upslope is more evident than that for albumin. For all experiments, the downslopes of the sulfate curve crossed over those of the labeled red cells, albumin, 58Co-EDTA and sucrose curves and then the water curve at around the peak. The downslopes of the [14C]salicylamide sulfate were similar to those for labeled water, suggesting rapid cellular influx and efflux of salicylamide sulfate. Quantitative evaluation with a barrier-limited space-variable transit-time model for rapidly equilibrating red cell and albumin binding accounted for the upslope effects on [14C]salicylamide sulfate behavior and demonstrated its relatively high liver cell permeability. Values for the unbound permeability surface area product (0.029 to 0.036 ml sec-1.gm-1) were not different, regardless of the presence or absence of red cells and albumin, and slightly exceeded values for hepatic blood flow (0.0186 +/- 0.0016 ml sec-1.gm-1). Comparable influx (0.083 to 0.14 sec-1) and efflux (0.10 to 0.18 sec-1) coefficients were obtained, suggesting rapid cellular entry and efflux of salicylamide sulfate.(ABSTRACT TRUNCATED AT 400 WORDS)
Albumins↗
Glycine conjugation activity of benzoic acid and its acinar localization in the perfused rat 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)
Animals↗
Methods for the quantitation of bromosulfophthalein and its glutathione conjugate in biological fluids.
This paper describes a solvent-gradient HPLC method which was developed for the quantitation of bromosulfophthalein (BSP) in erythrocyte and albumin containing (blood) perfusate samples, and of BSP and its glutathione conjugate (BSP-GSH) in bile samples obtained from rat liver perfusion experiments. Phenolphthalein was used as an internal standard. The cysteinyl-, N-acetylcysteinyl-, and cysteinylglycinyl-BSP conjugates did not interfere with the HPLC assay and cysteine, N-acetylcysteine, [3H]GSH, and [3H]GSSG eluted within the first 6 min postinjection onto the HPLC system. A spectrophotometric method was also developed for the rapid quantitation of BSP in blood perfusate; tracer [14C]urea was utilized as the internal standard. Although the spectrophotometric method was less sensitive than the HPLC method, good correlation was found to exist between the methods.
Animals↗
Formed and preformed metabolite excretion clearances in liver, a metabolite formation organ: studies on enalapril and enalaprilat in the single-pass and recirculating perfused rat liver.
Single-pass and recirculating rat liver perfusion studies were conducted with [14C]enalapril and [3H]enalaprilat, a precursor-product pair, and the data were modeled according to a physiological model to compare the different biliary clearances for the solely formed metabolite, [14C]enalaprilat, with that of preformed [3H]enalaprilat. With single-pass perfusion, the apparent extraction ratio (or biliary clearance) of formed [14C]enalaprilat was 15-fold the extraction ratio of preformed [3H]enalaprilat, an observation attributed to the presence of a barrier for cellular entry of the metabolite. Upon recirculation of bolus doses of [14C]enalapril and [3H]enalaprilat, the biliary clearance, estimated conventionally as metabolite excretion rate/midtime metabolite concentration, for formed [14C]enalaprilat was again 10- to 15-fold higher than the biliary clearance for preformed [3H]enalaprilat, but this decayed with perfusion time and gradually approached values for preformed [3H]enalaprilat. The decreasing biliary clearance of formed enalaprilat with recirculation was explained by the dual contribution of the circulating and intrahepatic metabolite (formed from circulating drug) to excretion. Physiological modeling predicted (i) an influx barrier (from blood to cell) at the sinusoidal membrane as the rate-limiting process in the overall removal of enalaprilat, (ii) a 15-fold greater extraction ratio or biliary clearance for formed [14C]enalaprilat over [3H]enalaprilat during single-pass perfusion, and (iii) the time-dependent and declining behaviour of the biliary clearance for formed [14C]enalaprilat during recirculation of the medium. In the absence of a direct knowledge of eliminating organs in vivo, this variable pattern for excretory clearance of the formed metabolite within the organ is indicative of a metabolite formation organ.
Animals↗
Combined recirculation of the rat liver and kidney: studies with enalapril and enalaprilat.
Combined recirculation of the rat liver (L) and kidney (IPK) at 10 ml min-1 per organ (LK) was developed to examine the hepatorenal handling of the precursor-metabolite pair: [14C]-enalapril and [3H]enalaprilat. Loading doses followed by constant infusion of [14C]enalapril and preformed [3H]enalaprilat to the reservoirs of the IPK or the LK preparation was used to achieve steady state conditions. In both organs, enalapril was mostly metabolized to its dicarboxylic acid metabolite, enalaprilat, which was excreted unchanged. At steady state, the fractional excretion for [14C]enalapril (FE = 0.45 to 0.48) and preformed [3H]enalaprilat (FE[pmi] = 1.1) were constant and similar for both the IPK and LK. The additivity of clearance was demonstrated in the LK preparation, namely, the total clearance of enalapril was the sum of its hepatic and renal clearances. However, the apparent fractional excretion for formed [14C]enalaprilat, FE(mi) and the apparent urinary clearance were time-dependent and higher than the corresponding values for preformed [3H]enalaprilat in both the IPK and LK. The FE(mi) and urinary clearance values further differed between the IPK and LK. Biliary clearance of formed vs. preformed enalaprilat displayed the same discrepant trends as observed for FE(mi) vs. FE(pmi) for the LK. These observations on the time-dependent and variable excretory clearance (urinary or biliary) of the formed metabolite vs. the constant, and much reduced, excretory clearance of the preformed metabolite are due to dual contributions to formed metabolite excretion: the nascently formed, intracellular metabolite which immediately underwent excretion and the formed metabolite which reentered the circulation, behaved as a preformed species. When data for the IPK and LK preparations were modeled with a physiological model with parameters previously reported for the L and IPK, all data, including metabolite excretory clearances, were well predicted. Model simulations revealed that the apparent FE(mi) differed between the LK and IPK preparations when the liver was present as an additional metabolite formation organ; the apparent excretory (urinary or biliary) clearance of the formed metabolite was further modulated by the volume of distribution of the metabolite, which altered levels of the formed, circulating metabolite.
Animals↗
Nonlinear protein binding and enzyme heterogeneity: effects on hepatic drug removal.
The kinetics of substrate removal by the liver and the resulting nonlinear changes in unbound fraction along the flow path at varying input drug concentrations were examined by a model simulation study. Specifically, we varied the binding association constant, KA, and the Michaelis-Menten constants (Km and Vmax) to examine the steady state drug removal (expressed as hepatic extraction ratio E) and changes in drug binding for (i) unienzyme systems and (ii) simple, parallel metabolic pathways; zonal metabolic heterogeneity was also added as a variable. At low KA, E declined with increasing input drug concentration, due primarily to saturation of enzymes; only small differences in binding were present across the liver. At high KA, a parabolic profile for E with concentration was observed; changes in unbound fraction between the inlet and the outlet of the liver followed in parallel fashion. Protein binding was the rate-determining step at low input drug concentrations, whereas enzyme saturation was the rate-controlling factor at high input drug concentration. Heterogeneous enzymic distribution modulated changes in unbound fraction within the liver and at the outlet. Despite marked changes in unbound fraction occurring within the liver for different enzymic distributions, the overall transhepatic differences were relatively small. We then investigated the logarithmic average unbound concentration and the length averaged concentration as estimates of substrate concentration in liver in the presence of nonlinear drug binding. Fitting of simulated data, with and without assigned random error (10%), to the Michaelis-Menten equation was performed; fitting was repeated for simulated data obtained with presence of a specific inhibitor of the high-affinity, anteriorly distributed pathway. Results were similar for both concentration terms: accurate estimates were obtained for anterior, high affinity pathways; an overestimation of parameters was observed for the lower affinity posteriorly distributed pathways. Improved estimations were found for posteriorly distributed pathways upon inhibition with specific inhibitors; with added random error, however, the improvement was much decreased. We applied the method for fitting of several sets of metabolic data obtained from rat liver perfusion studies performed with salicylamide (SAM) (i) without and (ii) with the presence of 2,6-dichloro-4-nitrophenol (DCNP), a SAM sulfation inhibitor. The fitted results showed that SAM sulfation was a high-affinity high-capacity pathway; SAM glucuronidation was of lower affinity but comparable capacity as the sulfation pathway, whereas SAM hydroxylation was of lower affinity and lower capacity.
Animals↗
Effect of protein binding on 4-methylumbelliferyl sulfate desulfation kinetics in perfused rat liver.
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)
Animals↗
Futile cycling between 4-methylumbelliferone and its conjugates in perfused rat liver.
Futile cycling between 4-methylumbelliferone and its sulfate and glucuronide conjugates was examined in the single-pass perfused rat liver preparation. The steady-state hepatic extraction ratio of 4-methylumbelliferone was found to be high (0.97) at a low input concentration of 0.005 mumol/L (tracer), with a net 4-methylumbelliferyl sulfate/4-methylumbelliferyl glucuronide ratio of about 5:1; at 63 mumol/L the steady-state extraction ratio had remained constant despite a shift from net sulfation to net glucuronidation. At higher input 4-methylumbelliferone concentrations, saturation was evidenced by a decreased steady-state extraction ratio and reduced net sulfation and net glucuronidation. Because 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide deconjugation would result in an intracellular accumulation of 4-methylumbelliferone, the phenomenon was monitored with a shift in tracer [3H]4-methylumbelliferone metabolism from sulfation to glucuronidation with increased intracellular 4-methylumbelliferone concentration. When 4-methylumbelliferyl sulfate (0 to 890 mumol/L) or 4-methylumbelliferyl glucuronide (0 to 460 mumol/L) was delivered simultaneously with tracer [3H]4-methylumbelliferone to the rat liver, notable desulfation of 4-methylumbelliferyl sulfate (18% to 38% rate in) but little deglucuronidation of 4-methylumbelliferyl glucuronide (1.2% to 2.1% rate in) was observed. With 4-methylumbelliferyl sulfate, 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were readily found as metabolites, whereas with 4-methylumbelliferyl glucuronide, levels of the metabolites, 4-methylumbelliferone and 4-methylumbelliferyl sulfate, were much reduced. 4-Methylumbelliferyl sulfate and not 4-methylumbelliferyl glucuronide shifted tracer [3H]4-methylumbelliferone metabolism from [3H]4-methylumbelliferyl sulfate to [3H]4-methylumbelliferyl glucuronide formation in a concentration-dependent fashion. The steady-state extraction ratio for 4-methylumbelliferyl sulfate (0.1 to 0.3) was comparatively higher than that for 4-methylumbelliferyl glucuronide (0.05), and it was found to increase with concentration, an observation explained by the nonlinear protein binding of 4-methylumbelliferyl sulfate. Biliary excretion rates for 4-methylumbelliferone and 4-methylumbelliferyl sulfate were proportional to their input or net formation rates, regardless of whether 4-methylumbelliferone, 4-methylumbelliferyl glucuronide or 4-methylumbelliferyl sulfate was administered. By contrast, the excretion rate of 4-methylumbelliferyl glucuronide when administered was only 1/25 the excretion of 4-methylumbelliferyl glucuronide formed from 4-methylumbelliferone and 4-methylumbelliferyl sulfate. The extent of choleresis paralleled the excretion patterns of preformed and formed 4-methylumbelliferyl glucuronide; bile flow was normal with 4-methylumbelliferyl glucuronide administration and was markedly enhanced with increased 4-methylumbelliferone or 4-methylumbelliferyl sulfate administration. The data suggest the presence of a transmembrane barrier for entry of 4-methylumbelliferyl glucuronide and not 4-methylumbelliferyl sulfate or 4-methylumbelliferone into hepatocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Stereoselectivity in glutathione conjugation and amidase-catalyzed hydrolysis of the 2-bromoisovalerylurea enantiomers in the single-pass perfused rat liver.
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)
Amidohydrolases↗
Kinetics of sequential metabolism. I. Formation and metabolism of oxazepam from nordiazepam and temazepam in the perfused murine liver.
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)
Animals↗