* Schering Plough Research Institute, P.O. Box 32, Lafayette, New Jersey 07848; and
Chemical Industry Institute of Toxicology, Research Triangle Park, North Carolina 27709
Received September 29, 1999; accepted January 24, 2000
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ABSTRACT |
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Key Words: blood ethanol concentrations; gastric and hepatic alcohol dehydrogenase; ethanol metabolism; PBPK modeling.
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INTRODUCTION |
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In addition, several factors have been shown to affect the bioavailability of ethanol that may influence toxicity. For example, administration of ethanol during fasting increases the bioavailability as compared to administration during the fed state (DiPadova et al., 1987; Gentry et al., 1992
). That is, blood ethanol concentrations (BEC) are higher in the fasted state than when ethanol is given with food. The concentration of ethanol administered can also alter the bioavailability. Humans who ingested 4% ethanol had higher BEC than those who ingested 10% ethanol (Sharma et al., 1993
). Similar effects of concentration have been found in rats (Roine et al., 1991
). The effect of the prandial state and concentration on bioavailability is, in part, mediated by altered absorption rates. Fasting and alcohol dilution are associated with faster gastric emptying (i.e., increased absorption rate), which in turn affects first-pass metabolic clearance (Gentry et al., 1994
).
Blood ethanol concentrations (BEC) in rats following a dose of 1.0 g/kg (Lim et al., 1993), and in humans following a dose of 0.15 g/kg (Julkunen et al., 1985
) illustrate the first-pass metabolism (FPM) of ethanol associated with oral ingestion. BEC are much higher following an intravenous (iv) administration when compared to an equivalent oral administration. Although FPM typically refers to loss due to hepatic metabolism, gastric metabolism has also been implicated in the decrease in bioavailability of ethanol. The terms gastric FPM (FPMG) and hepatic FPM (FPMH) have been introduced by several authors to provide the distinction (Lim et al., 1993
; Roine et al. 1991
). The relative contribution of each pathway in either rats or humans is not known with certainty and is the main focus of this paper.
Lim et al. (1993) proposed that the FPM of orally administered ethanol is due primarily to gastric ADH. Gastric ADH activity has been measured in rats (Lamboeuf et al., 1981; Caballeria et al., 1987
), mice (Algar et al., 1983
), and humans (Hempel and Peitruszko, 1975). Histamine2-blockers, such as cimetidine, inhibit gastric ADH activity in vitro (Palmer, 1987
) and also increase BEC, suggesting that metabolism by gastric ADH is at least partly responsible for the first-pass clearance (Caballeria et al., 1989
).
Despite the evidence regarding gastric FPM, other reports have implicated the liver as the primary site of FPM and also suggest dependency of bioavailability on the rate of ethanol absorption (Smith et al., 1992). Levitt and Levitt (1994) used a 2-compartment model developed for human males to illustrate that the FPM of ethanol was a result of hepatic metabolism and that gastric ADH did not contribute significantly to FPM. Their model also illustrated the dependency of first-pass clearance on the absorption rate of ethanol. The results from a pharmacokinetic model developed by Derr (1993) agree with those of Levitt and Levitt (1994).
FPM has been quantified by comparisons of the ratio of the blood area under the curve (AUC) following oral versus intravenous (iv) or intraperitoneal (ip) administration of ethanol (DiPadova et al., 1987). Another approach has been to quantify the total amount (mg) of ethanol absorbed following an oral versus an iv route of administration, with the difference being equal to the amount of FPM (Lim et al., 1993
; Roine et al., 1991
). Lim and coworkers (1993) administered ethanol by routes that bypassed the stomach (i.e., intraduodenal and intraportal), and found BEC equivalent to those obtained following an iv administration, thereby implicating the gastric mucosa as the primary site of FPM.
The reasons for the inconsistent results of the studies described above are not entirely clear. The use of invasive techniques may have been a factor. Animals were administered ethanol by the intraduodenal or intraportal route (Lim et al., 1993) or via isolated liver perfusions (Matsumoto et al., 1994
). These techniques require the use of anesthetics other than ethanol that can potentially influence results. In addition, the use of blood AUC may not accurately estimate bioavailability for chemicals whose metabolism can be saturated. When the rate of metabolism is first order, the bioavailability of a chemical is typically measured by blood AUCOral:AUVIV. However, when metabolism is pseudo-zero order, an increase in dose does not result in a proportional increase in blood AUC. That is, if the dose is doubled, the blood AUC is not necessarily doubled. Comparisons of AUC are appropriate only when BEC are low and the rate of metabolism is proportional to BEC. Ethanol metabolism is saturated at pharmacologically relevant doses (i.e., doses typically consumed by social drinkers and alcohol abusers). Therefore, blood AUCOral:AUCIV may not provide an accurate estimate of bioavailability under exposure scenarios of clinical relevance.
Physiologically based pharmacokinetic (PBPK) modeling (Pastino et al., 1997) has recently been used to characterize the disposition of ethanol. In contrast to classical pharmacokinetic methods, PBPK models take into consideration anatomical and physiological processes (tissue volumes and blood flows) as well as biochemical (metabolic rate constants) and physiochemical (partition coefficients) properties of the specific chemical (Clewell and Andersen, 1985
; Himmelstein and Lutz, 1979
). These characteristics allow for a more biologically based approach to quantifying FPM and bioavailability of ethanol than classical methods. A PBPK model can also be used to characterize the dosimetry of ethanol under a variety of conditions that may alter the bioavailability. The purpose of this study was to utilize a physiologically based pharmacokinetic (PBPK) model for ethanol to estimate the relative contributions of hepatic and gastric metabolic clearance to the oral bioavailability of ethanol in male rats.
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MATERIALS AND METHODS |
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The rate of change of ethanol in the stomach is a function of the rate of absorption and gastric metabolism, as given by:
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The typical tissue:blood partition coefficient, which normally describes the ratio of the chemical in the tissue relative to that in the blood, is not appropriate, because the partitioning of ethanol is dependent on the volume of the stomach and changes with time. Previous research estimated that the concentration of ethanol at the active site of gastric ADH is 4% of the concentration of ethanol in the stomach lumen (Smith et al., 1992). However, Pastino et al. (1996a) reported that the concentration of ethanol in the gastric mucosa exceeded 4% and changed over time relative to the amount in the stomach lumen. In the study by Pastino et al. (1996a), rats were administered 1.0 g/kg (16% w/v) ethanol orally and the ratio of ethanol in the gastric mucosa to the stomach lumen (i.e., stomach contents) was measured at several time points following the administration. This ratio is equivalent to PML (Equation 3
). A nonlinear regression analysis of these data presented by Pastino et al. (1996a) provided the following equation:
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The experimental data used in this model development were BEC obtained from rat tail blood. Previous research demonstrated that during periods of rising and declining ethanol levels, the concentration in the tail lagged behind the arterial, jugular or femoral vein blood (Levitt et al., 1994). Concentrations in the tail blood are not equivalent to the pooled venous blood concentration at early time points after bolus dosing. The equation must take into account the rate of transfer into the tail blood. Thus, the concentration of ethanol in the tail blood (CVTail) was described by:
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The blood flows and tissue volumes for each compartment (Table 1) were obtained from the report prepared by the International Life Sciences Institute, Risk Science Institute, the United States Environmental Protection Agency on "Physiological Parameter Values for PBPK Models" (International Life Science Institute, 1994). The ethanol partition coefficients for rats were determined by Kaneko et al. (1994). The kidney:blood partition coefficient was used for the rapidly perfused compartment, and the skeletal muscle:blood partition coefficient was used for the slowly perfused compartment. The absorption and metabolic rate constants were determined as outlined below.
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Rate Constants for Metabolism by Alcohol Dehydrogenase in Male Rats.
Mammalian ADH exists in multiple molecular forms (Agarwal and Goedde, 1990; Bosron and Li, 1986
; Kedishvili et al., 1995
). There are several isozymes of rat ADH, specifically ADH1, ADH2, and ADH3. ADH3 is primarily responsible for ethanol metabolism in the liver whereas ADH1 is responsible for ethanol metabolism in the stomach (Julia et al., 1987
). While each enzyme is comprised of two active subunits and require zinc and NAD+ for metabolic activity, the kinetic properties of each isozyme differ. For example, the isoelectric points are 5.1 and 8.258.4 for ADH1 and ADH3, respectively (Julia et al., 1987
). In addition, the KM for ethanol oxidation differs significantly. The potential for each isozyme to contribute to the in vivo elimination of ethanol is therefore different. In the interests of model parsimony, only a single Michaelis-Menten pathway was described in the gastric and hepatic compartments. Attempting to describe multiple pathways in each compartment would not be a useful exercise given the extent of the data available for parameter estimation. The metabolic pathways described in the current model can thus be thought of as representing the average metabolic behavior of the enzymes capable of metabolizing ethanol.
The hepatic and gastric ADH KMs utilized in the PBPK model were experimentally determined by Caballeria et al. (1989; 23 mg/L and 18,400 mg/L, respectively). The remaining rate constants were optimized against experimental BEC and included KTail, VmaxH, KaS and VmaxG (Table 2). KTail and VmaxH were optimized against BEC following an iv administration of 250 mg/kg [Caballeria et al., 1989; numerical data was kindly provided by Joan Caballeria (personal communication)] and 1.0 g/kg (Lim et al., 1993
). The model was coded to account for the multi-step iv infusion rate used by Lim et al. (1993). In this study, 50% of the dose (1.0 g/kg) was administered in the first 15 min, 25% given over the next 15 min, 12.5% given over the following 90 min, and the final 12.5% over the last 240 min. The total infusion time was 6 h.
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Calculation of Gastric First Pass Metabolism of Ethanol.
The first-pass clearance of orally administered ethanol results from metabolism by gastric ADH, which occurs prior to absorption from the gastrointestinal (GI) tract into the liver, and metabolism by the liver through the first pass prior to absorption into the systemic circulation. The amount of gastric ethanol metabolism (AMG; mg) was calculated using the PBPK model, through integration of the Michaelis-Menten expression, for metabolism by gastric ADH (Equation 2). Gastric FPM, represented as a percentage of the administered dose, was then calculated by:
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Calculation of Hepatic First-Pass Metabolism of Ethanol.
In order to calculate FPMH, a distinction between the two sources of hepatic ethanol metabolism was made. The assumptions of the PBPK model are that the liver is well mixed and delivery of ethanol to the liver is blood-flow limited. Ethanol enters the liver from the GI tract and as recirculating ethanol. Regardless of how ethanol reaches the liver, it is metabolized by a saturable system having Michaelis-Menten kinetics characterized by Vmax and KM. The total rate of hepatic ADH metabolism (RAMT; mg/h) is described by:
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Given that the liver is assumed to be well mixed, ethanol entering the liver by one route competes for metabolism of ethanol entering by another route. The rate of metabolism by each pathway (i.e., RAMR L and RAMGI L) can therefore be described by the equation for competitive inhibition (York, 1997). In describing the rate of metabolism of ethanol entering the liver from the GI tract, the substrate concentration is the concentration of ethanol in the liver resulting from newly absorbed ethanol from the GI tract, and the concentration of the inhibitor is the concentration of recirculating ethanol. The inhibitor affinity constant is the hepatic ADH KMH for ethanol oxidation. The rate of metabolism of ethanol entering the liver from the GI tract is estimated by:
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Calculation of the Oral Bioavailability of Ethanol.
The bioavailability, represented as a percentage of the total dose administered (BIO), was calculated as:
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RESULTS |
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The optimizations provided accurate simulations of the experimental data, with the possible exception of the BEC, following oral administration of 1000 mg/kg reported in Lim et al. (1993; Fig. 3, bottom panel, circles). It is unclear why the PBPK model overpredicted the Lim et al. (1993) data but not the Roine et al. (1991) data. The only differences between the experimental conditions in these studies was the body weight of the animals. The dose, concentration, and strain of rats were the same in both studies. The PBPK model accounted for the body weights when simulating each respective data set, as illustrated by the differences in the PBPK model simulations (Fig. 3
, bottom panel). Although the specific ages of the animals were not specified in each study, the animals used in the Roine et al. (1991) study were purchased as adults whereas the animals used in the Lim et al. (1993) study were purchased as weanlings. It is possible that the age difference may have affected the rates of hepatic metabolism and therefore the BEC (Seitz et al., 1992
). The simulations in Figures 2 and 3
were obtained using a single hepatic ADH Vmax and did not account for possible age-dependent differences in hepatic metabolism.
The experimental data used for the optimization procedures were tail BEC obtained following both oral and iv administration. Previous research found that, during periods of rising and declining ethanol levels, tail BEC lag behind concentrations in the arterial, jugular, and femoral vein blood in rats (Levitt et al., 1994). This is due to the low blood perfusion to tissue water ratio in the tail. The simulations in Figure 4 illustrate the discrepancy between pooled venous BEC and tail BEC following a bolus oral administration. At earlier time points, tail BEC are lower than pooled venous BEC. However, these concentrations eventually equilibrate.
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DISCUSSION |
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In calculating FPMH, the PBPK model provided a distinction between metabolism of incoming ethanol from the GI tract and metabolism of recirculating ethanol. Under the assumptions of a well-stirred liver, Michaelis-Menten elimination kinetics, and flow-limited delivery of ethanol, the ethanol entering the liver from the GI tract necessarily competes for metabolism with ethanol that has entered the liver through recirculation. When the duration of absorption from the GI tract is long with respect to the time it takes the blood to recirculate, the calculation of FPMH must take into account the competition with recirculating ethanol. Accordingly, at least some of the ethanol will be metabolized during the first pass through the liver, even when concentrations in the blood are high. Thus, the bioavailability should never reach 100%, as was illustrated by the PBPK model.
In addition, because hepatic and gastric metabolism were described as saturable processes, the predicted bioavailability should be dose-dependent. As the amount of ethanol administered increases, the capacity of the liver to metabolize it, relative to the amount presented to the liver, decreases, and more of the ethanol escapes metabolism during the first pass. An increase in bioavailability is therefore expected with an increase in the dose, based on the kinetic characteristics of gastric and hepatic ADH.
The calculated FPMH of ethanol is higher than previously found (Levitt and Levitt, 1994) and is probably due to the respective definition and the method for calculating FPM. The traditional definition of FPM is the removal of chemicals before entrance into the systemic circulation, typically by metabolism in the gut or liver (Rozman and Klaassen, 1996
). However, Levitt and Levitt (1994) defined hepatic FPM as:
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ACKNOWLEDGMENTS |
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NOTES |
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