Biomedical subjects

K S Pang

Publications and source records attributed to K S Pang.

At least 37 records · Page 2Linked to original sources

PubMed1998

The modified dipeptide, enalapril, an angiotensin-converting enzyme inhibitor, is transported by the rat liver organic anion transport protein.

Oatp1, the organic anion transport polypeptide, is an integral membrane protein cloned from rat liver that mediates the uptake of various organic anions such as bromosulfophthalein (BSP) and taurocholate (TCA). Recent studies by others revealed that the thrombin inhibitor, CRC 220, a modified dipeptide, was transported by oatp1. The present study was designed to examine whether another modified peptide, enalapril, an angiotensin-converting enzyme inhibitor, was also a substrate. Transport was studied with enalapril (1 to 800 micromol/L, with [3H]enalapril) in a HeLa cell line stably transfected with oatp1-cDNA under the regulation of a Zn2+-inducible promoter. Noninduced transfected cells (without zinc) that did not express oatp1 failed to take up enalapril. In contrast, cells expressing oatp1 transported enalapril, estrone sulfate (E1S), taurolithocholic acid sulfate (TLCAS), and the glutathione conjugate of BSP (BSPGSH). Uptake of enalapril by oatp1 at 37 degreesC was substantially higher than that at 4 degreesC. The rate at 37 degreesC (uptake rates for induced - noninduced, transfected cells) was linear over 5 minutes and was concentration-dependent, characterized by a Km of 214 +/- 67 micromol/L and a Vmax of 0.51 +/- 0.15 nmol/min/mg protein. Enalapril uptake was inhibited competitively by BSP (at 1, 5, 10, and 50 micromol/L) and TCA (at 5, 25, and 100 micromol/L) with inhibition constants (Ki) of 2 and 32 micromol/L, respectively. The metabolite enalaprilat was, however, not transported by oatp1. That oatp1 is not a general transporter of anionic compounds was further shown by the lack of transport of harmol sulfate, benzoate, and hippurate. These observations attest to the role of oatp1 as a specific transporter for at least two classes of pharmacologically important peptides.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Application of the dispersion model for description of the outflow dilution profiles of noneliminated reference indicators in rat liver perfusion studies.

The dispersion model (DM) is a stochastic model describing the distribution of blood-borne substances within organ vascular beds. It is based on assumptions of concurrent convective and random-walk (pseudodiffusive) movements in the direction of flow, and is characterized by the mean transit time (t) and the dispersion number (inverse Peclet number), DN. The model is used with either closed (reflective) boundary conditions at the inflow and the outflow point (Danckwerts conditions) or a closed condition at the inflow and an open (transparent) condition at the outflow (mixed conditions). The appropriateness of DM was assessed with outflow data from single-pass perfused rat liver multiple indicator dilution (MID) experiments, with varying lengths of the inflow and outflow catheters. The studies were performed by injection, of bolus doses of 51Crlabeled red blood cells (vascular indicator), 125I-labeled albumin and [14C] sucrose (interstitual indicators), and [3H]2O (whole tissue indicator) into the portal vein at a perfusion rate of 12 ml/ min. The outflow profiles based on the DM were convolved with the transport function of the catheters, then fitted to the data. A fairly good fit was obtained for most of the MID curve, with the exception of the late-in-time data (prolonged tail) beyond 3 x [symbol: see text]. The fitted DNS were found to differ among the indicators, and not with the length of the inflow and outflow catheters. But the differences disappeared when a delay parameter, t0 = 4.1 +/- 0.7 sec (x +/- SD), was included as an additional fitted parameter for all of the indicators except water. Using the short catheters, the average DN for the model with delay was 0.31 +/- 0.13 for closed and 0.22 +/- 0.07 for mixed boundary conditions, for all reference indicators. Mean transit times and the variances of the fitted distributions were always smaller than the experimental ones (on average, by 6.8 +/- 3.7% and 58 +/- 19%, respectively). In conclusion, the DM is a reasonable descriptor of dispersion for the early-in-time data and not the late-in-time data. The existence of a common DN for all noneliminated reference indicators suggests that intrahepatic dispersion depends only on the geometry of the vasculature rather than the diffusional processes. The role of the nonsinusoidal ("large") vessels can be partly represented by a simple delay.

Animals↗

PubMed1998

Hepatic uptake of hippurate: a multiple-indicator dilution, perfused rat liver study.

The hepatic transport of hippuric acid (HA), a glycine-conjugated metabolite of benzoic acid that exhibits only modest plasma albumin binding (binding association constant of 2.1 x 10(3) M-1), was studied in the single-pass perfused rat liver (12 ml/min), using the multiple indicator dilution (MID) technique. The venous recovery of [3H]HA on portal venous injection of a MID dose containing a mixture of a set of noneliminated reference indicators and [3H]HA revealed a survival fraction of unity, corroborating the lack of disappearance of bulk HA from plasma. When the outflow recovery was fitted to the barrier-limited model of Goresky et al. (C.A. Goresky, G. G. Bach, and B. E. Nadeau. J. Clin. Invest. 52: 991-1009, 1973), the derived influx (P(in)S) and efflux (P(out)S) permeability-surface area products were found to be dependent on the concentration of HA (1-930 microM); P(in)S and P(out)S were approximately 3.5 times the plasma flow rate at low HA concentration, but decreased with increasing HA concentration. All values, however, greatly exceeded the expected contribution from passive diffusion, because the equilibrium distribution ratio of chloroform to buffer for HA was extremely low (0.0001 at pH 7.4). The tissue equilibrium partition coefficient (P(in)/P(out), or ratio of influx to efflux rate constants, k1/k-1) was less than unity and decreased with concentration. The optimized apparent Michaelis-Menten constant and maximal velocity were 182 +/- 60 microM and 12 +/- 4 nmol.s-1.g-1, respectively, for influx and 390 +/- 190 microM and 29 +/- 13 nmol.s-1.g-1, respectively, for efflux. In the presence of L-lactate (20 mM), however, P(in)S for the uptake of HA (174 +/- 3 microM) was reduced. Benzoic acid (10-873 microM) was also effective in reducing hepatic uptake of HA (5.3 +/- 0.9 microM). These interactions suggest that MCT2, the monocarboxylate transporter that mediates the hepatic uptake of lactate and other monocarboxylic acids, may be involved in HA transport.

Animals↗

PubMed1998

Hepatic uptake of bromosulfophthalein-glutathione in perfused Eisai hyperbilirubinemic mutant rat liver: a multiple-indicator dilution study.

The hepatocellular uptake of the glutathione conjugate of bromosulfophthalein (BSPGSH) was examined in Eisai hyperbilirubinemic rats (EHBR; originating from Sprague-Dawley rats), which lacked the ATP-dependent canalicular transport for non-bile acid organic anions, a trend common to other mutant rat strains (TR- and GY, originating from Wistar rats). Single-pass perfused rat liver experiments were conducted with BSPGSH (26-257 microM) using the multiple indicator dilution technique. The steady-state extraction ratio of BSPGSH was close to zero due to lack of biliary excretion. After the introduction of a bolus dose containing vascular (51Cr-labeled red blood cells), interstitial (125I-labeled albumin and [14C]sucrose) and cellular space (D2O) indicators and [3H]BSPGSH into the portal vein, the outflow dilution profile of [3H]BSPGSH was found to display a protracted declining profile (tailing) at low input BSPGSH concentrations; the tail disappeared at higher BSPGSH concentrations. When data were fitted with the barrier-limited model of Goresky as used previously for BSPGSH for the Sprague-Dawley rat (SDR), model fitting was found to evoke an additional "deep pool" within the hepatocyte to account for the "tail" component. The deep pool became evident for the EHBR because biliary excretion of BSPGSH was absent and the rate of return from the deep pool was slow. The concentration of BSPGSH within the deep pool was estimated to be 12 +/- 8 times that in the cytosol. The binding of BSPGSH to EHBR S9 (effective binding concentration of 53 microM and a binding association constant KA of 2.4 x 10(4) M-1), however, was found to be lower than that of SDR S9 and could not account for the late-in-time data. The influx permeability-surface area product was concentration dependent and decreased from 0.27 to 0.01 ml.sec-1.g-1 with increasing BSPGSH concentration; the throughput component, or the portion of the dose that goes through the liver without entering the hepatocyte, increased with increasing concentration. The trends were characteristic of carrier-mediated transport and were similar to those found for the uptake of BSPGSH in SDR.

Animals↗

PubMed1998

Intracellular and not intraluminal esterolysis of enalapril in kidney. Studies with the single pass perfused nonfiltering rat kidney.

Two possible sites of renal metabolism exist: intracellular, by enzymes within the peritubular cells, and intraluminal, by ecto-enzymes embedded on the brush border membrane. The esterolysis of enalapril to its dicarboxylate metabolite, enalaprilat, was studied in the isolated perfused, nonfiltering rat kidney preparation (NFK) and compared with that observed for the isolated perfused rat kidney (IPK) to ascertain the site of metabolic conversion. For the NFK, filtration was obliterated with the high oncotic pressure (8% bovine serum albumin in plasma) and ligation of the ureter, thus preventing enalapril from reaching intraluminal sites by filtration. The steady-state renal plasma clearance of enalapril in the NFK was 2.0 ml/min/g, a value similar to that (2.1 ml/min/g) observed previously for the IPK. The rate of appearance of enalaprilat, the metabolite, in venous plasma for the NFK (30 +/- 3% of the input rate of enalapril) was also comparable with that for the IPK (27 +/- 4%). Further, identification of the site of enalapril metabolism (cellular or luminal) was aided by simulations based on physiological models and parameters obtained previously on the renal handling of enalapril and enalaprilat. These parameters were optimized to match closely the experimental observations. The predicted total and metabolic renal clearances for the IPK or for the NFK were similar for both the "cellular model" and "luminal model": in both instances, values for the NFK were 59-65% of those for the IPK. By contrast, predictions for the venous output rate of enalaprilat (as a percent of the input rate of enalapril) were different: the "cellular model" predicted no change in value between the NFK and the IPK, whereas metabolite appearance was greatly magnified for the NFK (289% that of the IPK) with luminal metabolism. The lack of difference in venous outflow of enalaprilat for the NFK and IPK was more congruent with the notion of intracellular and not intraluminal esterolysis of enalapril.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1998

Hepatic clearance models: comparison of the dispersion and Goresky models in outflow profiles from multiple indicator dilution rat liver studies.

The multiple indicator dilution (MID) technique is often used for investigation of the kinetic behavior of substrates and metabolites in eliminating organs. The present study was a systematic comparison of the utility of the Goresky model (GM) (a structural model) and the mixed-boundary dispersion model (DM) (a stochastic model) in the interpretation of influx, efflux, and removal (sequestration) coefficients, with data generated from rat liver-perfusion/MID studies. Although the GM and the DM are equivalent in their descriptions of membrane transport, they differ in their classifications of the dispersion of blood-borne elements. For the DM, the dispersion is an inverse Gaussian distribution of vascular transit times; for the GM, it is accounted for by the dispersion observed among noneliminated reference indicators (e.g. labeled red blood cells, albumin, sucrose, and H2O) or the derived reference. In this study, previously published rat liver-perfusion/MID data obtained for the glutathione conjugate of bromosulfophthalein and hippuric acid, compounds that exhibit saturable carrier-mediated transport, with the GM were reanalyzed with the two-compartment DM. When the fitted values for volume and transfer coefficients were compared, good correlation was found between the fitted vascular volume for the DM and the vascular volume for the reference indicator for the GM. The influx coefficients were generally similar between the models, but improved correspondence was observed when the DM was modified to include the large-vessel transit time. In contrast, the efflux and sequestration coefficients obtained for the DM did not correspond well to those from the GM. The disagreement was due, in part, to differences in the interpretation of the late-in-time component of the reference transit time distribution curve, which was not described well by the DM. Consequently, the residence time distribution and the relative dispersion were underestimated by the DM.

Animals↗

PubMed1997

Organ clearance concepts: new perspectives on old principles.

The removal capacity of an eliminating organ by metabolism and/or excretion is often expressed as its clearance. Metabolic and excretory clearances are considered to be mutually independent, and the sum of these constitute the whole organ clearance. The influence of metabolism on estimates of the excretory clearance and vice versa was examined for the liver and kidney with physiologically based models. Mass transfer first-order rate equations describing transport and removal were derived. Upon inversion of the matrices originating from the coefficients of these equations, the area under the curve (AUC) and clearance (dose/AUC) were obtained with the liver or kidney as the eliminating organ. A more complex solution was found to exist for the kidney since glomerular filtration, secretion; reabsorption, and intrarenal metabolism were present. To ascertain the effect of excretion on estimates of the metabolic clearance as well as the effect of metabolism on estimates of the excretory clearance, intrinsic clearances for excretion or metabolism were set to zero. Clearance values were found to be altered when alternate pathways were present. Whereas excretory clearance estimates were consistently reduced in the presence of metabolism, metabolic clearance estimates were affected differentially by excretion and varied according to the site of metabolism. Excretion reduced metabolic clearance estimates when metabolism occurred intracellularly. If metabolism occurred intraluminally (e.g., on the renal brush border or luminal membrane), the metabolic clearance estimate could become higher since the substrate was made available to the enzymes following its excretion. As expected, these changes depended on the relative magnitudes of the intrinsic clearances for metabolism and excretion. The above theory was applied to the elimination of enalapril which is both metabolized and excreted by the perfused rat liver and kidney preparations. Data obtained in these studies were consistent with a set of published physiologic parameters denoting transfer and intrinsic clearances. Perturbations on clearance estimates were studied by setting the metabolic/excretory intrinsic clearance to zero, then to some finite value. In liver, the avid hepatocellular metabolism of enalapril reduced biliary clearance by 73%. For the kidney, the fractional excretion (FE or unbound excretory clearance/glomerular filtration rate) was decreased modestly (from 0.64 to 0.44) with intracellular esterolysis, whereas if metabolism had occurred intraluminally, FE would have been significantly decreased (from 1.8 to 0.45). Simulation results show clearly that clearance estimates are affected by the presence of alternate removal pathways, and question the well-established principle the metabolic and excretory clearance estimates are independent of each other.

Animals↗

PubMed1997

First-pass effect: significance of the intestine for absorption and metabolism.

The occurrence of low systemic availability due to significant metabolism or poor absorption of orally administered drugs has been well recognized. Three rate controlling factors affecting the oral absorption: unstirred water layer, membrane limitation, or flow limitation, have been identified. These are much affected by the physicochemical properties of the drug: pKA, water/lipid solubility, structural mimicry to endogenous substrates for transport proteins, and the physiology of the GI tract. Drug metabolizing enzymes are found to be present in the intestine, albeit the content is lower than that found in liver. The presence of pre-absorptive versus post-absorptive intestinal metabolism is presently discussed in experimental sets of data with luminal and systemic administration of the drugs in the vascularly perfused rat small intestine preparation. The effect of the anterior anatomical placement of the intestine and its contribution to metabolism, in relation to that for the liver, has been examined in our laboratory by the perfused intestine-liver preparation. The effect of concentration and flow have been studied and general principles governing drug absorption and metabolism in the intestine and the subsequent effects on the liver have been discussed.

Animals↗

PubMed1997

Effect of flow on first-pass metabolism of drugs: single pass studies on 4-methylumbelliferone conjugation in the serially perfused rat intestine and liver preparations.

The vascularly perfused rat intestine and liver preparations were used to examine the effect of flow (8 and 10 ml/min) on the sequential metabolism of 4-methylumbelliferone (4MU), which forms primarily the glucuronide conjugate (4MUG) in intestine and the sulfate conjugate (4MUS) in liver at low input concentrations of 4MU. In this system, a constant tracer concentration of [3H]4MU was delivered systemically at 8 or 10 ml/min to the perfused rat small intestine preparation; the portal venous outflow perfusate at 8 and 10 ml/min was collected at steady state, reoxygenated and in tum delivered to the perfused rat liver preparation from a second rat donor. The intestinal extraction ratio and formation of 4MUG were decreased from 0.57 +/- 0.07 to 0.49 +/- 0.06 and 42 +/- 5 to 36 +/- 4% input rate, respectively, upon increasing the flow rate from 8 to 10 ml/min (P < .05). These decreases were the result of the reduction in transit time with increasing flows. In contrast, hepatic 4MU conjugation was increased (from 40 +/- 7% to 48 +/- 6% input rate to intestine) upon increasing the flow rate from 8 and 10 ml/min (P < .05), attributed primarily to increased formation of the major metabolite, 4 MUS, in liver (from 35 +/- 9% to 39 +/- 9% input rate to intestine). The unusual observation on increased hepatic metabolite formation with increasing flow could be rationalized. With increased flow to the serially perfused organs, there was an increased supply of substrate to the liver, the posterior organ, because of a faster intestinal transit time. Decreased intestinal metabolism (formation of 4MUG) at increased flow was compensated by increased hepatic metabolism (formation of 4MUS), albeit attenuated because of a faster hepatic transit time. The proportions of total 4MU conjugates formed (4MUG + 4MUS) across the intestine and liver remained constant at both flow rates. Hence, a rather constant overall extraction ratio (0.98 +/- 0.004 and 0.97 +/- 0.005, P > .05) existed across the two organs. The results demonstrate that the intestine, the anterior organ, plays a regulatory role on substrate supply to the posterior organ, the liver. With an increase in flow, the contribution of the intestine will decrease, whereas the contribution of the liver will increase in the overall first-pass metabolism.

Animals↗

PubMed1997

Inhibition of glutathione conjugation by glutathione analogues in the perfused rat liver. Effect of esterification on the potency of gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine.

To assess the role of GST's (glutathione S-transferases) in the (de)toxification of their substrates, an in vivo active inhibitor based on the structure of glutathione (GSH), gamma-L-glutamyl-alpha-(D-2-aminoadipyl)-N-2-heptylamine monoethyl ester (Et-R-Hep), was developed. To increase its effectivity, analogues esterified with alkyl chains of varying lengths and one diesterified derivative (DiEt-R-Hep) were synthesized. The unesterified analogue, R-Hep, was also tested. Their isoenzyme selectivity was characterized using purified rat GST isoenzymes. Furthermore, the extent of inhibition of the GSH conjugation of (RS)-2-bromoisovalerylurea (BIU) was evaluated in rat liver cytosol, isolated hepatocytes, and in liver perfusions. All compounds inhibited Alpha- (1-1 and 2-2) more effectively than Mu (3-3 and 4-4) class GSTs; Pi-(5-5) and Theta (7-7) classes were minimally inhibited. The unesterified R-Hep was the most effective inhibitor towards purified isoenzymes; its Ki value towards GST 3-3 (S-BIU as substrate) was 27 microM. The mono ethyl ester derivative, Et-R-Hep (Ki 270 microM for 3-3), was the most potent inhibitor in hepatocytes and in the perfused liver: 50 microM inhibited the conjugation of (S)-BIU by 50%. Longer ester chains or diesterification did not increase the inhibitory potency; R-Hep had less inhibitory activity. In all systems, only the (S)-enantiomer of BIU, which is conjugated mainly by Alpha class GSTs, was inhibited, confirming Alpha isoenzyme selective inhibition.

Animals↗

PubMed1996

Lack of glutathione conjugation of melphalan in the isolated in situ liver perfusion in humans.

Tumor cell resistance against melphalan (LPAM) has been associated with increased cellular reduced glutathione (GSH) levels and glutathione S-transferase activity. Therefore, GSH conjugation of LPAM has been hypothesized to be a key factor in tumor cell resistance. In the present study, we evaluated GSH conjugation of LPAM by the perfused liver in patients with colorectal cancer metastases undergoing a Phase II study of isolated liver perfusion as well as in the rat. To evaluate whether LPAM-GSH conjugates were synthesized in the rat in vivo, LPAM was infused i.v. at a rate of 2.0 micromol/kg/min. In bile samples obtained during the infusion, two major GSH conjugates were identified by mass spectrometry: mono-hydroxy-mono-GSH-LPAM and di-GSH-LPAM. The maximum biliary excretion rate of these two conjugates accounted for only 1.3% of the LPAM infusion rate. In bile or perfusate samples from patients treated for 60 min initially with 0.3 mM LPAM in the perfusion medium via isolated liver perfusion (200 mg LPAM in approximately 2 liters perfusion medium), none of the above-mentioned conjugates were detected. When comparable rat liver perfusions were performed initially with 66 microM or 0.66 mM LPAM in the perfusion medium, bile samples did contain GSH-LPAM conjugates; the cumulative biliary excretion of the two conjugates amounted to 0.4 and 0.2% of the LPAM dose, respectively. These data suggest that both in rats and humans, hepatic GSH conjugation plays a very minor (if any) role in the elimination of LPAM and, therefore, that modulation of GSH levels is unlikely to affect the rate of elimination of this drug.

Animals↗

PubMed1996

Dynamics of arterial and portal venous flow interactions in perfused rat liver: an intravital microscopic study.

Intravital epifluorescent microscopy was used to quantitate microvascular parameters in the single-pass, dually perfused rat liver preparation. Livers perfused via the hepatic artery (HA) and portal vein (PV) at physiological pressures and perfusion rates responded to vasoactive agents and exhibited the HA buffer response. The distribution of arterial blood was found to be highly heterogeneous, whereas PV flow was distributed uniformly. The intrasinusoidal velocity of fluorescein isothiocyanate (FITC)-labeled red blood cells (RBCs) arriving from the HA was higher than that for RBCs arriving from the PV, indicating a shorter transit time for the arterially delivered FITC-RBCs. Experiments on livers perfused simultaneously via the HA and retrogradely via the hepatic vein revealed the presence of arteriovenous shunts, with some of the arterially delivered FITC-RBCs reaching the terminal hepatic venules via direct channels without traversing the sinusoidal bed. In livers perfused portally only, changes in PV flow rate (from 8 to 20 ml/min) produced small changes in perfusion pressure but large changes in vascular diameters, while portal pressure and transit time of portal blood remained relatively constant. In experiments designed to identify the location of hepatic vascular resistance, it was observed that hepatic venular diameters measured in the preparation under identical pressure and flow conditions were greater during retrograde than during prograde perfusion, suggesting that the site of hepatic vascular resistance is presinusoidal or sinusoidal.

Animals↗

PubMed1996

Uptake of sulfate conjugates by isolated rat hepatocytes.

The uptake of estrone sulfate (E1S; 1 to 400 microM), harmol sulfate (HS; 5 to 900 microM), and 4-methylumbelliferyl sulfate (4MUS; 5 to 1000 microM) was investigated in isolated rat hepatocytes in the presence or absence of inhibitors. Uptake of all of the sulfate conjugates was rapid and exhibited saturation kinetics, best characterized by saturable and nonsaturable (linear transmembrane clearance) transport systems. The KM's were: 16 +/- 6, 123 +/- 28, and 64 +/- 6 microM for E1S, HS, and 4MUS, respectively, with corresponding Vmax's of 0.85 +/- 0.56, 0.48 +/- 14, and 0.42 +/- 0.07 nmol/min/10(6) cells. The nonsaturable uptake clearances, which displayed concentration-independent uptake, were 3 +/- 2, 1 +/- 0.1, 0.5 +/- 0.1 microliter/min/10(6) cells, respectively. Uptake of E1S was inhibited by ouabain (1 mM) and replacement of sodium by choline, whereas HS was insensitive to the addition or substitution. Uptake of both E1S and HS was significantly reduced by metabolic inhibitors (antimycin A, 2.7 microM, rotenone, 30 microM, and KCN, 2 mM) and temperature reduction (from 37 to 27 degrees C). 4,4'-Diisothiocyanostilbene-2-2-'disulfonic acid (2 mM), an inhibitor of anion transport, reduced E1S and HS uptake; E1S uptake was also reduced by HS. HS uptake by both saturable and nonsaturable transport components was depressed by 4MUS (300 microM); the apparent KM was increased by 83% while the Vmax remained unaltered, and the nonsaturable component was decreased by 48%. The data strongly suggest that multiple pathways exist for the uptake of E1S, HS, and 4MUS. E1S uptake is sodium-dependent, requires energy, and is inhibited by anions such as 4,4'diisothiocyanostilbene-2-2'-disulfonic acid and other sulfate conjugates. HS uptake, while being energy dependent, is not sodium dependent, and is inhibited by 4MUS in a competitive fashion. At least one of these pathways is shared.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1996

Sequestered endoplasmic reticulum space for sequential metabolism of salicylamide. Coupling of hydroxylation and glucuronidation.

The metabolic disposition of simultaneously delivered [14C]salicylamide (SAM) (100 microM) and a tracer concentration of its hydroxylated metabolite [3H]gentisamide (GAM) was studied with single-pass followed by recirculating rat liver perfusion (10 ml/min). The use of dual radiolabeling of precursor-product pairs in single-pass and recirculating perfusions allowed for characterization of the differential metabolism of preformed [3H]GAM and formed [14C]GAM, which arose in situ in the liver with [14C]SAM single-pass perfusion, and the behavior of circulating [14C]GAM, which behaved as a preformed species in recirculation. In both modes of perfusion, [14C]SAM was mainly sequentially metabolized to [14C]GAM-5-glucuronide, whereas [3H]GAM predominantly formed [3H]GAM-5-sulfate. The steady-state and time-averaged clearances of SAM were identical and approached the value of flow, yielding a high hepatic extraction ratio (E = 0.98). The apparent extraction ratio of formed GAM [E(mi) = 0.96] was greater than that of the preformed species [E(pmi) approximately 0.7]. Because the coupling of (SAM) oxidation and (GAM) glucuronidation was a plausible explanation for the observation, a novel physiological pharmacokinetic model was developed to interpret the data. In this model, the liver was divided into three zonal units, within which acinar distribution of enzymatic activities was considered, namely periportal sulfation, evenly distributed glucuronidation, and perivenous hydroxylation. Each zonal region was subdivided into extracellular, cytosolic, and endoplasmic reticulum compartments, with cytosolic (sulfotransferases) and microsomal (cytochromes P-450 and UDP-glucuronosyltransferase) enzymes being segregated intracellularly into the cytosolic compartment and endoplasmic reticulum compartment, respectively. The simulations provided a good prediction of the present experimental data as well as previously obtained data with increasing SAM concentration and retrograde flow and supported the contention that SAM oxidation and GAM glucuronidation are coupled.

Animals↗

PubMed1995

Carrier-mediated uptake and excretion of bromosulfophthalein-glutathione in perfused rat liver: a multiple indicator dilution study.

The hepatic removal of the glutathione conjugate of bromosulfophthalein (BSPGSH) was studied in the single-pass perfused rat liver with the multiple indicator dilution (MID) technique against various background concentrations of BSPGSH (20 to 214 mumol/L) over which nonlinear binding to both plasma (albumin) and tissue proteins with two classes of binding sites was found. A bolus containing 51Cr-labeled red blood cell (a vascular reference), [125I]albumin and [14C]sucrose (large and small molecular weight interstitial references, respectively), D2O (a cellular space reference), and [3H]BSPGSH was injected into the portal vein during steady-state. The eliminated fraction of dose, obtained by subtracting the survival fraction of [3H]BSPGSH in plasma from one, corresponded to the steady state extraction ratio (E) with bulk data, which declined from 0.74 +/- 0.04 to 0.27 +/- 0.01 with concentration. The major portion of the tracer outflow profile was a throughput component, which is the proportion of tracer that did not enter liver cells during its transit through the liver. The influx, efflux, and sequestration coefficients, evaluated with previously developed barrier-limited models, provided the corresponding influx (k1), efflux (k-1) and excretion (kseq) rate constants. Concentration-dependent influx (Vmax = 83 nmol min-1 g-1 and Km = 3.7 mumol/L), efflux (Vmax = 15 nmol min-1 g-1 and Km = 1.8 mumol/L), and excretion (Vmax = 94 nmol min-1 g-1 and Km = 1.8 mumol/L) were obtained for BSPGSH, when Km values are expressed in terms of the unbound concentrations. In these calculations, the observed unbound tissue concentration was not used for estimation of the Vmax and Km for efflux and excretion because of overestimation, because of the presence of highly concentrated BSPGSH in ductular elements present in liver homogenates; rather, the unbound tissue concentration was calculated from the influx, efflux, and removal rate coefficients. Because of carrier-mediated entry, the unbound tissue concentration does not equal the unbound plasma concentration, and kinetic parameters for BSPGSH excretion could be alternately estimated when the rate of excretion or net rate of loss of BSPGSH from plasma was regressed against the estimated tissue unbound concentration. This yielded a Vmax of 97 nmol min-1 g-1 and a Km of 3.6 mumol/L, values similar to those obtained from MID.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

An understanding of flow- and diffusion-limited vs. carrier-mediated hepatic transport: a simulation study.

Concentration-dependent changes in the hepatic extraction ratio E and tissue accumulation of drugs were examined in a simulation study, wherein plasma protein binding, flow, and mode of entry were altered. A tubular flow model that described carrier-mediated (influx: Kml = 20 microM, Vmax1 = 1000 nmol min-1; efflux, Km2 = 200 microM, Vmax2 = 250 nmol min-1), flow-limited (influx clearance CLin = efflux clearance CLef = 50 ml min-1), or diffusion-limited (CLin = CLef = 0.1 ml min-1) hepatocytic entry was employed; drug removal was solely via biliary excretion (Km3 = 100 microM, Vmax3 = 1500 nmol min-1). Other parameter space and the combination of carrier-mediated transport and passive diffusion were also explored. Increased plasma protein binding reduced the hepatic extraction of the substrate, and in some instances, constituted the rate-controlling factor, especially at lower input concentrations for which tighter binding existed. Increased flow rate also brought about a reduction in E, affecting E almost inversely when values of E were low (e.g., for the diffusion-limited case or at higher input concentration). Tissue accumulation patterns and the apparent tissue distribution equilibrium ratio, i.e., tissue to plasma unbound concentration ratio Kp, differed among the systems. The behavior of Kp may be used as an identifier for the mode of drug transport: A declining (concave-down) Kp curve or a parabolic Kp that approached unity with input concentration (Cln) is associated with carrier-mediated entry; a rising Kp curve that approaches unity with Cln suggests flow limitation; and a waning concave-up Kp curve of very low magnitude represents diffusion limitation. Since the unbound tissue concentration (Ct) differs from the logarithmic average of the unbound input and output concentrations in plasma (Cu) for carrier-mediated and diffusion-limited systems, excretion parameters may be obtained only upon fitting of the overall excretion rate vs. Ct in the Michaelis-Menten equation; whereas when data are fitted with Cu, the rate-limiting step, influx, or deviations of influx, efflux, and excretion, will be obtained. When Ct equals Cu, as in flow-limited systems, accurate excretion parameters will be provided with the fitting of data against either Ct or Cu.

Carrier Proteins↗

PubMed1995

Concentration-dependent metabolism of diazepam in mouse liver.

Previous mouse liver studies with diazepam (DZ), N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generated via NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precursors of OZ. In microsomal studies, the Kms and Vmaxs, corrected for binding to microsomal proteins, were 34 microM and 3.6 nmole/min per mg and 239 microM and 18 nmole/min per mg, respectively, for N-demethylation and C3-hydroxylation of DZ. The Kms and Vmaxs for N-demethylation and C3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 microM and 2.5 nmole/min per mg and 311 microM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 microM, the extraction ratio of DZ (E[DZ]) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disproportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and the in vitro enzymatic parameters provided a poor in vitro-organ correlation. The E[DZ], appearance rates of the metabolites, and the extraction ratio of formed NZ (E[NZ, DZ]) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters improved the correlations and identified NZ as the major contributor of OZ. Saturation of DZ N-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates used in vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poor in vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics.

Animals↗

PubMed1995

Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver.

The relation between the rate of glutathione (GSH) conjugation and hepatic GSH content was studied in the rat in vivo and the in situ single-pass-perfused rat liver preparation with bromosulfophthalein (BSP) as the model substrate. The biliary excretion of the BSP-GSH conjugate and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during liver perfusions with BSP-containing perfusion medium. Rats were pretreated with single or multiple doses of buthionine sulfoximine, an inhibitor of the de novo synthesis of GSH. Surprisingly, the excretion of the BSP-GSH conjugate was sustained at a high rate, despite a virtually complete depletion of hepatic GSH, both in the rat in vivo as well as in the perfused rat liver. The results indicate that GSH was still available for conjugation with BSP after apparent depletion of the hepatic GSH pool, presumably because of a residual de novo synthesis of GSH in the liver. Despite the multiple pretreatment with buthionine sulfoximine, the de novo GSH synthesis was sufficient to sustain a high rate of GSH conjugation of BSP. The cosubstrate-Km for GSH conjugation of BSP in the liver was estimated to be very small (approximately 0.3 mumol/g): the excretion rate of the BSP-GSH conjugate was only impaired at minimal hepatic GSH levels.

Animals↗