The influence of enzyme localization and blood flow on intestinal
elimination was evaluated in rats. Phenol was administered vascularly
(~1400 and 2500 µg) and luminally (intrajejunal bolus doses of
~100 and 1000 µg) to the recirculating in situ perfused intestine. The portal effluent and the reservoir were sampled. The intestinal extraction ratios for phenol at the low and high vascular doses were (mean ± S.D., n = 3)
0.09 ± 0.02 and 0.11 ± 0.01, respectively. The perfusion
flow rate was also varied from 5 to 12 ml/min at a vascular dose of
~2500 µg of phenol. The organ clearance at the lowest flow rate
significantly exceeded those at the higher flow rates. The presence of
a diffusional barrier at the mucosa-serosa interface was suggested. The
calculated mean diffusional clearance of phenol was 1.11 ml/min.
Sulfation was the predominant metabolic pathway after vascular
administration of phenol. After luminal dosing, the intestinal
intrinsic clearances of phenol at the low and high doses were 7.29 ± 1.39 (n = 4) and 3.55 ± 1.16 ml/min
(n = 3), respectively, indicating saturation at the
higher dose. Moreover, there was a decrease in the area under the curve
ratio (metabolite/phenol) at the high luminal dose. Luminal
administration, in general, produced greater glucuronidation. These
data and STELLA simulations suggest that enzyme localization at both
the cellular and tissue levels has a significant influence on
intestinal metabolism.
 |
Introduction |
Physiological
models of hepatic extraction have been used quite successfully to
predict in vivo clearance from in vitro data. The well stirred model
(Pang and Rowland, 1977
) is most frequently used, owing in part to its
simplicity. However, a similar application of the well stirred model to
the intestine was less successful (Thummel et al., 1997). A possible
reason for its failure might be the polarized expression of
drug-metabolizing enzymes within the intestine (Chowdhury et al., 1985;
Watkins, 1997). Most enzymes involved in intestinal elimination, such
as the UDP-glucuronosyltransferases (UGTs2),
cytochrome P450 3A (CYP3A), and efflux pumps such as
P-glycoprotein, are expressed in the epithelial cells (Chowdhury
et al., 1985; Watkins, 1997). On the other hand, the vasculature
("serosa") is devoid of significant enzymatic activity. This seems
to refute the premise of the intestine as a well stirred organ.
Because the intestine can receive compounds luminally and vascularly,
elimination could occur both during and after absorption, phenomena
called pre- and postabsorptive intestinal elimination (Routledge and
Shand, 1979). The polarized intestinal tissue might allow drug
administered perorally to have better access to these eliminating sites
than that administered vascularly, leading to greater intestinal
extraction and possible qualitative differences in metabolite kinetics,
as well. Thus, intestinal clearance is potentially a function of
intrinsic clearance as well as the ability of the drug to diffuse to
the epithelial eliminating site, i.e., its diffusional clearance. An
assessment of these factors led Gwilt and colleagues (1988) to model
the intestine as three compartments: the lumen, the metabolically
active mucosa, and the blood in equilibrium with the gut tissue, which
can be considered the serosa (Gwilt et al., 1988). These authors
proposed that differences in pre- and postabsorptive intestinal
elimination could arise due to a "diffusional barrier" at the
mucosa-serosa interface. A diffusional barrier is not necessarily a
membranous barrier per se but could be an increased diffusional path
length as a consequence of the polarized localization of intestinal enzymes.
Phenol, the model substrate used in the present study, is extensively
metabolized by the rat gut (Cassidy and Houston, 1984). It has a simple
biotransformational fate, being largely converted to a sulfate and a
glucuronide with essentially all of the metabolites being renally
eliminated (Capel et al., 1972). Phenol is glucuronidated by members of
the UGT1A family (Inoue et al., 1999) and sulfated by the phenol
sulfotransferases (Oddy et al., 1997). The UGTs show considerable
intestinal expression in rats (Mojarrabi and Mackenzie, 1998; Inoue et
al., 1999; Tukey and Strassburg, 2000). The UGTs display a decreasing
expressional gradient in epithelial cells from the villus to the crypt
(Chowdhury et al., 1985) and are localized between the nuclear and
apical membrane of the epithelial cell (Inoue et al., 1999). The active
site of the UGTs is found within the endoplasmic reticulum (ER) lumen
(Burchell and Coughtrie, 1989). The phenol sulfotransferases (PSTs) are
also found in the intestinal mucosa (Dunn and Klaasen, 1998) and are
cytosolic enzymes (Burchell and Coughtrie, 1997). Thus, both enzymes
display some form of tissue and cellular localization.
The objectives of the present study were to establish a relationship
between intestinal organ clearance and its putative determinants, namely, diffusional clearance, intrinsic clearance, and intestinal blood flow, and to evaluate the role of enzyme localization on intestinal metabolism. Phenol was administered vascularly and luminally
to the recirculating in situ perfused intestine (IPI) and the IPI with
luminal dosing. The influence of tissue and cellular localization of
drug-metabolizing enzymes on intestinal organ clearance and metabolite
kinetics was studied.
| |
Materials and Methods |
Phenol, phenyl glucuronide, p-cresol, sodium
pentobarbital, glucose, bovine serum albumin (25% in Tyrode's
solution), and sulfatase (type H-I from Helix pomatia) were
all obtained from Sigma-Aldrich (St. Louis, MO). Dextran T-40 was
obtained from Amersham Biosciences (Piscataway, NJ). All other
chemicals were reagent grade or better. Packed human red blood cells
were obtained from the American Red Cross (St. Paul, MN).
Vascular Dosing: The Recirculating IPI.
Male Sprague-Dawley rats, 250 to 275 g (Harlan, Indianapolis, IN),
were used. Surgery was conducted with minor modification of a procedure
described previously (Hirayama et al., 1989). The perfusate was
Krebs-Henseleit bicarbonate buffer (pH 7.4), bovine serum albumin,
Dextran T-40, and washed human red blood cells in appropriate
proportions (Hirayama et al., 1989), and the superior mesenteric
arterial perfusate flow rate was 7.5 ml/min (Davies and Morris, 1993).
The effluent perfusate from the portal vein was returned to the
reservoir. A small incision was made in the jejunum to accommodate a
glass cannula, and luminal fluid was collected during the course of the
experiment. The viability of the perfused organs was continuously
monitored by means of blood pressure at the superior mesenteric artery
(SMA) and pO2 in the effluent portal perfusate.
Phenol was administered at vascular doses of 1400 and 2500 µg to the
reservoir. Samples (300 µl) were taken from the reservoir and portal
vein at 3, 8, 15, 30, 45, and 60 min. In addition, a reservoir sample
taken before the start of the experiment was used to calculate the
dose. Samples were centrifuged at 13,000g for 2 min (Fisher
Microcentrifuge, model 250B; Fisher Scientific, Pittsburgh, PA). The
supernatant was stored at 
20°C until further analysis.
Luminal Dosing: In Situ Perfused Intestine with Luminal Dosing.
Surgery was performed on anesthetized male Sprague-Dawley rats to
initiate a blank IPI. The blank vascular perfusate was recirculated until the preparation was stabilized at 37°C. The jejunum was ligated
2 to 3 cm from the ligament of Trietz, and a loose ligature was placed
15 cm distal to it. A small incision was made in the jejunum at this
point, and a metal feeding tube was secured in it by tightening the
ligature around it. The hub of the feeding tube was attached to a glass
syringe containing the dose, and this was introduced as a bolus at time
0. Samples (300 µl) were taken from the reservoir at 0, 10, 25, 55, and 70 min and from the portal effluent at 2, 5, 10, 30, 55, and 70 min. Sample handling was as described above. At the end of the
experiment, the jejunal loop was flushed with air and saline. The
luminal fluid collected was used to determine the amount in the lumen
at the end of the experiment and, when subtracted from the original
dose, gave the amount absorbed during the course of the experiment. Two
luminal doses of phenol (100 and 1000 µg) were administered.
Analytical Methods.
Phenol samples were analyzed by HPLC with fluorescence detection. The
HPLC setup consisted of a Beckman Gold 126 system (Beckman Coulter,
Inc., Fullerton, CA) with Gold Nouveau integration software (version
1.6; Beckman Coulter) and a RF 530 fluorescence detector (Shimadzu
Corp., Kyoto, Japan). A reversed-phase C18
Supelcosil (4.6 × 250 mm; 5 µ particle size; Supelco,
Bellefonte, PA) column was used. The mobile phase consisted of buffer
and acetonitrile (75:25 parts by volume) delivered at a flow rate of 1 ml/min with isocratic elution. The buffer was 0.05 M
KH2PO4 (Fisher Scientific) with 5 mM tetrabutylammonium phosphate (Regis Technologies, Inc., Morton Grove, IL) as an ion-pairing agent. The pH of the buffer was
adjusted to 5.5 with 1% phosphoric acid. The excitation and emission
wavelengths were 260 and 305 nm, respectively.
Standard curves of 1 to 80 µg/ml and 2.5 to 100 µg/ml were
constructed for phenol and phenyl glucuronide, respectively. The internal standard was p-cresol (50 µg/ml). The retention
times for phenyl glucuronide, phenol, and p-cresol were 3.9, 8.3, and 13.7 min, respectively. A phenyl sulfate standard was not
commercially available; hence, an enzymatic incubation with sulfatase
was used. An aliquot of 200 µl of the sample supernatant was split
into two 100-µl portions. One part was assayed for phenol and the
glucuronide as described above. The other 100 µl was incubated with
40 µl of sulfatase (5000 U/ml) for 16 h in a shaking water bath
at 37°C. The difference between the phenol concentration before and
after sulfatase incubation yielded the concentration of phenyl sulfate. Because a phenyl glucuronide standard was available, it could be shown
that the sulfatase preparations were devoid of glucuronidase activity.
There was no decrease in phenyl glucuronide concentrations in perfusate
samples undergoing sulfatase incubation (data not shown).
The HPLC assay for phenol and phenyl glucuronide was validated for
inter- and intraday precision (% coefficient of variation, % CV) and
accuracy. These were found to be within acceptable limits, with a % CV
and bias of less than 10%, except for the lowest concentration of the
phenyl glucuronide, which had a % CV of approximately 15% (Kothare,
2001).
Data Analysis.
The actual vascular dose administered was calculated by multiplying the
initial concentration of drug in the reservoir by the volume of the
reservoir. Due to the surgical setup, the gut was the only eliminating
organ in the "circuit". Thus, the postabsorptive gut clearance,
CLgw, org, could be calculated by
|
(1)
|
where AUCres is the area under the
concentration-time curve of the drug in the reservoir from 0 to 
(Rowland et al., 1973). The AUCres from 0 to the
last experimental time point, t, was calculated by the
linear trapezoidal rule. The concentration-time profile was fit
exponentially with KaleidaGraph (version 3.5; Abelbeck/Synergy,
Reading, PA) to obtain a terminal slope, 
. The AUC from the last
time point to infinity was calculated by dividing the concentration at
the last time point by . The two partial AUCs were added to obtain
the AUC from 0 to infinity. The intestinal extraction ratio,
Egw, was calculated as
|
(2)
|
where Qres was the perfusate flow rate.
After luminal dosing, the preabsorptive clearance was calculated based
on an equation developed from the model in Fig.
1. In the figure,
Cres, Cgw, and
Cgw, in were the concentrations of the drug in
the reservoir, the gut wall, and entering the gut wall, respectively.
Cpv was the concentration of the drug leaving the
gut wall. The rate of drug entry from the lumen to the gut wall was
given by dX/dt. The preabsorptive clearance calculated was the
intrinsic clearance (CLgw) of the gut. The
concentration entering the gut wall, Cgw,in, was
considered equivalent to the concentration leaving the reservoir,
Cres. The concentration leaving the gut wall,
Cpv, was assumed to be equivalent to the
concentration within the gut wall, Cgw. This
assumption is justified for phenol because it has a very high free
fraction (approximately 86%) in a similar perfusion medium (Ballinger
et al., 1995); hence, the free drug may be considered to be in a rapid
equilibrium between the tissue water and the perfusate.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the model used
to calculate intestinal clearance after luminal dosing.
In the figure,Cres is the concentration of drug in the
reservoir; Cgw is the concentration of drug in the gut
wall; Cgw,in is the concentration of drug entering the gut
wall; Cpv is the concentration of drug in the portal vein;
Qres is the perfusate flow rate; CLgw is the
intestinal intrinsic clearance. X represents the dose, dX/dt is the
rate of drug entry from the lumen to the gut wall, and  represents a
sampling site.
|
|
A mass balance equation for the rate of change of drug amount with time
within the gut wall (dgw/dt) was obtained:
Substituting the assumptions of the model in the above equation:
where Vgw, the volume of gut wall, was
taken as 11 ml/250 g rat (Davies and Morris, 1993).
Multiplying both sides of the equation by dt and integrating with
respect to time from 0 to t:
where AUCx,t was the AUC in
compartment "x " from time 0 to t, calculated
by the linear trapezoidal rule; X' = [Xt X0] was the amount of drug absorbed
from the gut lumen from time 0 to t;
Cpv,t was the drug concentration in the
portal vein at time "t " minutes. The intestinal
intrinsic clearance (CLgw) was obtained by
rearranging the above equation to obtain:
|
(3)
|
All statistical analyses involved analysis of variance and were
conducted with StatView (version 4.01; SAS Institute, Cary, NC).
Vascular Dosing of Phenol with Varying Vascular Flow Rates.
Experiments were conducted at flow rates of 5 (n = 3),
10 (n = 2), and 12 (n = 2) ml/min with
2500 µg vascular doses of phenol. In addition, data were used from
the aforementioned study in vascularly dosed rats with a flow rate of
7.5 ml/min. Flow rates of less than 5 ml/min were attempted but were
unsuccessful because the mesenteric vessels appeared to collapse.
Theoretical Evaluation of Intestinal Clearance.
The data analyses described in the preceding section regarded the gut
wall as a single compartment, and the pre- and postabsorptive intestinal clearances calculated were the intrinsic clearance and organ
clearance by the gut, respectively. To get a clearer understanding of
the physiological determinants of intestinal clearance, a more rigorous
theoretical analysis was done; the model employed is shown in Fig.
2. The gut wall was divided into two
compartments, the eliminating mucosa and the non-eliminating serosa. A
diffusional clearance, CLdiff, governed mass
transfer between the two compartments. The intrinsic clearance from the mucosa, CLgw, was responsible for intestinal
elimination. Both vascular and luminal dosing cases were evaluated. The
detailed derivations are provided in Appendix I.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
The diffusional model of intestinal
clearance.
The amounts of the drug in the reservoir, serosa, and mucosa are given
by Xr, Xs, and Xm, respectively.
The perfusate flow rate is Q. represents a sampling site. The
diffusional clearance between the serosa and mucosa is
CLdiff. The intrinsic clearance of the drug by the gut is
CLgw. After luminal dosing, X represents the dose and dX/dt
is the rate of drug entry from the lumen into the mucosa.
|
|
Based on this analysis, after vascular dosing of drug, the
fraction escaping gut wall clearance, fg, was
given by:
|
(4)
|
where
| Q |
|
= |
|
intestinal blood flow |
| CLdiff |
|
= |
|
diffusional clearance of the drug between the serosa and mucosa |
| CLgw |
|
= |
|
intrinsic clearance by the gut |
Thus, the fraction escaping gut wall clearance,
fg (and therefore, Egw and
the intestinal organ clearance, CLgw,org from eq. 2), is a complex function of the blood flow to the organ, the diffusional clearance, and the intrinsic clearance of the drug. It is
interesting to consider three limiting conditions of eq. 4.
Case 1: If there is no diffusional barrier
(CLdiff 
CLgw)
|
(4a)
|
This is the situation where there is a perfusion rate-limited
distribution of the drug, and the diffusional clearance has no effect
on the extraction ratio. This equation is essentially the same as that
for the well stirred model of the liver.
Case 2: If there is a diffusional barrier at the mucosa-serosa
interface (CLdiff 
Q)
|
(4b)
|
In such a case, there is a diffusion rate-limited distribution
of drug and no extraction results.
Case 3: For a drug with high intrinsic clearance and poor diffusion
(CLgw CLdiff)
|
(4c)
|
In this case, the extent of extraction is determined by the
diffusional clearance between the mucosal and serosal spaces.
Also based on the diffusional model in Fig. 2, after luminal
dosing, the AUC measured to infinity in any compartment yielded the
intrinsic clearance, CLgw (Appendix IB)
|
(5)
|
The above model highlights the potential importance of
diffusional barriers in intestinal extraction. Cases 2 and 3 clearly display situations where a large difference between pre- and
postabsorptive elimination might result from the existence of a
diffusional barrier at the serosa-mucosa interface.
Diffusional Clearance of Phenol.
After vascular dosing in the diffusional model, the AUC to infinity in
the reservoir is given by eq. A-8 (in Appendix IA). A rearrangement of
that equation was used to calculate the diffusional clearance of phenol
|
(6)
|
where
| CLdiff |
|
= |
|
diffusional clearance of the drug |
| D |
|
= |
|
vascular dose of drug |
| Q |
|
= |
|
blood flow rate |
| AUCr |
|
= |
|
AUC to infinity of parent in the reservoir |
| CLgw |
|
= |
|
intrinsic clearance by the gut (previously calculated from the luminal
dosing of phenol). |
STELLA Simulations.
To get a better understanding of the metabolite kinetics of phenol,
simulations were done with STELLA simulation software (Macintosh
version 4.0; HPS Systems, Hanover, NH). An initial model (Model I) was
built that assumed the gut to be a single compartment. Because this did
not satisfactorily simulate the experimental results, a more complex
model (Model II) was built further dividing the gut wall based on the
cellular localization of the conjugating
enzymes. The schematics of the models
used are provided in Figs. 3 and 4.
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 4.
A schematic of STELLA Model II dividing the
gut wall based on the cellular compartmentalization of intestinal
enzymes.
|
|
| |
Results |
Viability.
The perfusate pressure at the SMA and the portal
pO2 were used to assess viability. For both
drugs, a standard deviation of not more than 5% from the mean was seen
during the course of the experiment (Kothare, 2001), indicating
viability of the preparations. In the studies with different flow
rates, the SMA pressure showed a rising trend with increasing flow
rate, indicating an increasing resistance within the blood vessels. The
pO2 levels also increased with flow, indicating
more anaerobic conditions presumably due to a shorter residence time.
Vascular Dosing.
Table 1 summarizes the postabsorptive
intestinal metabolism of phenol. A low "postabsorptive" extraction
ratio was obtained after vascular dosing for phenol, and there was no
statistical difference between the extraction ratios obtained at the
high and low vascular doses. Figure 5
shows the concentration-time profiles of phenol at the low vascular
dose. The phenol concentration in the reservoir declined in a biphasic
manner. The higher vascular dose of phenol revealed a similar pattern
(Kothare, 2001).
View this table:
[in this window]
[in a new window]
|
TABLE 1
Extraction ratios and intestinal organ clearances after vascular dosing
of phenol
Data are expressed as mean ± S.D. of n = 3.
|
|
The two metabolites of phenol showed different shapes in the
concentration-time curves (Fig. 5). The sulfate appeared early and then
reached an asymptotic value. The glucuronide, on the other hand,
appeared later and ascended slowly. An analysis of the luminal
secretions showed that neither phenol nor the conjugates were secreted
to a large extent (<1% of dose) (Kothare, 2001).
At the high vascular dose of phenol, the portal AUC ratio
(sulfate/phenol), 0.30 ± 0.06, was statistically greater
(p < 0.05) than the portal AUC ratio
(glucuronide/phenol), 0.13 ± 0.02. Similarly, at the low vascular
dose, the portal AUC (sulfate/phenol), with a value of 0.29 ± 0.14, statistically exceeded the portal AUC ratio (glucuronide/phenol)
at 0.02 ± 0.02. This confirmed sulfation as the predominant
metabolic pathway after vascular dosing.
Luminal Dosing.
In sharp contrast to the vascular dosing case, the low luminal dose of
phenol was more extensively metabolized, as evidenced by comparing the
concentration-time profiles (Fig. 6) and
the value of the AUC ratio (metabolite/phenol) from each route to those
in Fig. 5. At the low luminal dose of phenol, the intestinal intrinsic
clearance approached the perfusate flow rate (Table 2). Glucuronidation was the predominant
metabolic pathway in the majority of rats, with the glucuronide levels
exceeding those of the sulfate at most time points. At the low luminal
dose of phenol, the portal AUC ratio (glucuronide/phenol) was 1.18 ± 0.52 whereas the portal AUC ratio (sulfate/phenol) was 0.74 ± 0.35. The AUC ratio (metabolite/phenol) overall exceeded that after vascular dosing, indicating greater metabolism.
The distinct metabolic profiles seen with vascular dosing of phenol
were repeated with the luminal dose (Fig. 6). Phenyl sulfate again
reached an asymptote. In contrast, phenyl glucuronide continued its
ascending trend even at later time points when most of the phenol (the
assumed driving force) was all but gone. This trend was repeated in the
concentration-time profiles at the higher luminal dose of phenol as
well (Kothare, 2001). However, at the high luminal dose, the intrinsic
intestinal clearance was reduced by half, indicating saturation of
metabolism (Table 2). At the higher luminal dose of phenol, the portal
AUC ratio (glucuronide/phenol) was 0.18 ± 0.05 whereas the portal
AUC ratio (sulfate/phenol) was 0.15 ± 0.10. Both routes showed a
statistically lower AUC ratio (metabolite/phenol) at the higher luminal
dose, indicating that saturation was occurring in both pathways.
Based on eq. 6, the mean diffusional clearance
(CLdiff) of phenol could be calculated. Because
CLgw was determined from luminal dosing and the
AUCr was determined after vascular dosing, it was necessary to use the doses from the two routes of administration that
generated similar "effective" concentrations at the enzyme site.
Keeping this in mind, the high vascular dose of phenol (~2000 µg),
when divided by the reservoir volume (200 ml), yielded an effective
concentration of 10 µg/ml. The low luminal dose (~100 µg), when
divided by the rat intestinal volume of ~11 ml for a 250 g rat
(Davies and Morris, 1993), yielded a concentration of ~9 µg/ml. The
initial effective concentrations generated by the high vascular dose
and the low luminal dose were, therefore, comparable. The diffusional
clearance (CLdiff) of phenol was 1.11 ml/min, which was much smaller than the perfusion flow rate. This is similar to
Case 2 (eq. 4b) and is a possible explanation for the negligible postabsorptive extraction seen.
Effect of Flow Rate on Clearance.
The results of varying the flow rate are summarized in Table
3. A Fisher's post hoc analysis revealed
that the postabsorptive extraction ratio, Egw, at
the lowest flow rate (5 ml/min) was significantly greater than that at
each of the higher flow rates (p < 0.05). The
gut extraction ratios at each of the higher flow rates (7.5-12 ml/min)
were statistically similar to each other. The intestinal organ
clearance, CLgw,org, at 5 ml/min was also significantly lower than that at each of the higher flow rates. No
obvious trends in the phenol concentration-time profiles were seen
(Kothare, 2001). The AUC ratio (metabolite/phenol) at each of the flow
rates showed that sulfation remained the major metabolic pathway at
each of the flow rates and there were no obvious flow-dependent trends
in the overall formation of the metabolite (Kothare, 2001). However, a
comparison of the glucuronide concentration-time profiles from the
reservoir samples (Fig. 7) revealed that
there was a greater lag time in the appearance of the glucuronide at 5 ml/min compared with each of the higher flow rates.
STELLA Modeling.
There is no evidence in the literature to suggest sequential metabolism
for either of the conjugates of phenol in the rat. Therefore, in a
recirculating system, as long as substrate is present the conjugates
should display a biphasic accumulation curve (an initial rapid
formation phase followed by subsequent slower accumulation phase). This
was not observed for either of the metabolite concentration-time
profiles. In addition, concentration-time profiles of the two
conjugates showed distinctly different shapes.
STELLA simulation software was used to get a better mechanistic
understanding of the metabolite kinetics. Simulations were conducted
for the vascular and luminal dosing case; however, only the luminal
dosing case is discussed here. The equations used to set up the models
will be provided by the authors upon request (also published in
Kothare, 2001). An initial model, termed "Model I" (Fig. 3),
assumed that formation was the rate-limiting step in the appearance of
the conjugates in the perfusate. As expected, the simulated reservoir
concentration-time profile yielded a biphasic ascending curve (Fig.
8) in contrast to the observed reservoir concentration-time profile (Fig. 6). Therefore, the more complicated Model II (Fig. 4) was developed. The gut wall was divided into cytosolic and ER compartments. The simulated reservoir
concentration-time profiles now generated (Fig.
9) were more consistent with the experimentally observed profiles in Fig. 6. Also, perhaps more interestingly, the concentration-time profile of the glucuronide in the
various compartments (Fig. 10) showed a
significant accumulation of the glucuronide within the ER lumen. Thus,
the simulations suggested a rate-limiting step involved in the efflux
of the glucuronide as well as a metabolic recycling of the sulfate.
View larger version (12K):
[in this window]
[in a new window]
|
Fig. 10.
Simulated concentration-time profile of the
glucuronide in the reservoir and ER compartment based on Model II.
Note that the concentrations in the reservoir and the ER are on
different y-axis scales.
|
|
| |
Discussion |
This study aimed to experimentally evaluate the role of enzyme
localization in pre- and postabsorptive intestinal elimination. A
thorough theoretical assessment of a diffusional model of intestinal elimination was conducted. Alternate models, including those based on
the segregation of intestinal blood flow, have also been evaluated and
will be published in due course.
The postabsorptive intestinal extraction ratio of phenol was very
small. It was indeed much lower than the values reported in the
literature after intraduodenal dosing of phenol (Cassidy and Houston,
1984). After vascular dosing, the drug apparently has limited access to
the apically expressed metabolizing enzymes. From a diffusional
perspective, the effective path length of a vascularly administered
drug molecule to the enzyme site would be magnified severalfold due to
the tortuosity of its pathway from the vasculature to the villus tip.
As a result, its "effective diffusivity" after vascular
administration could be much lower than that after luminal administration.
A theoretical analysis revealed that after vascular dosing, the
fraction escaping gut wall metabolism (as well as the intestinal organ
clearance) was a complex function of intestinal blood flow, intrinsic
clearance of the drug by the gut, and the diffusional clearance of the
drug between the serosa and the mucosa. The calculated diffusional
clearances of phenol were much smaller than the intestinal blood flow.
In such a situation, the theoretical model predicted that the fraction
escaping gut wall metabolism would be unity. Indeed, the experimental
results showed negligible postabsorptive intestinal extraction ratios.
In other words, there was a diffusional barrier to the metabolism of
the vascularly administered drug at the mucosa-serosa interface.
Because the vascular blood flow is also an important determinant of
intestinal clearance, the effect of changing vascular flow rate on
intestinal organ clearance was evaluated. The lowest flow rate of 5 ml/min yielded statistically greater extraction than the higher flow
rates, presumably due to a greater residence time within the tissue,
but the extraction ratio was still very low. Because the diffusional
clearance calculated was much smaller than the vascular flow rate, Case
2 (eq. 4b) of the theoretical model was applicable and predicted that
the entire dose would escape gut wall metabolism. As predicted, changes
in blood flow had little effect on the extraction ratio. The lowest
flow rate was associated with a greater lag time in the appearance of
the glucuronide compared with the higher flow rates.
In marked contrast to the vascular dosing case, after the low luminal
dose of phenol, the concentrations of phenol approached zero by the end
of the experiment. Moreover, the AUC ratios (metabolite/phenol) after
luminal dosing exceeded those observed after vascular dosing. In
addition, the intrinsic gut clearance at the low dose approached the
intestinal blood flow, qualifying phenol as a high clearance drug
(Cassidy and Houston, 1984).
Similar differences between luminal and vascular dosing were reported
earlier from this lab (Wen et al., 1999) with 6-aminocarbovir (6AC), a
prodrug of the carbocyclic nucleoside, carbovir. The conversion of 6AC
to carbovir is carried out by adenosine deaminase, which is also
mucosally expressed (Chinsky et al., 1990). Both the 6AC and the phenol
examples clearly refute the premise of the intestine as a well stirred
organ; hence, models of intestinal extraction contingent on a diffusion
rate-limited distribution of drug seem more appropriate. From a
practical standpoint, the loss of this heterogeneity in disruptive
systems like cell layers, enterocytes, and microsomes probably
contributes to their frequent failure to predict in vivo intestinal clearance.
Two aspects of the metabolite kinetics of phenol were notable
the
distinct shapes of each of the metabolite curves and the route
dependence of the formation of the primary metabolite. Because the
literature does not suggest sequential metabolism of either conjugate,
these curves may be more reflective of enzyme localization at the
cellular level. In theory, the plateau observed with the sulfate
reservoir concentration profile could indicate either a saturation in
its formation or a metabolic recycling to the parent. Sulfate ions were
constantly supplied to the intestine by way of the Krebs-Henseleit
buffer in the vascular perfusate; hence, a depletion of the cosubstrate
seems unlikely. Intracellular metabolic recycling to the parent phenol
might explain the plateauing of the sulfate curve. In fact, the
reservoir sulfate profiles showed a double peak effect, which would be
in keeping with such a recycling.
After luminal administration, the glucuronide concentrations continued
to rise even as phenol concentrations (the presumed driving force)
approached zero. Furthermore, there was a decrease in AUC ratio
(metabolite/phenol) and intrinsic clearance in going from the low to
high luminal dose. This may indicate a saturable process being involved
in the release of the glucuronide from the cell.
STELLA simulation software was used to gain a better understanding of
the metabolite kinetics of phenol. Model I assumed formation rate-limited metabolite kinetics in a "closed" recirculating
system. Predictably, the metabolites showed biphasic ascending
concentration curves, unlike the experimentally observed results.
Thus, the more complicated Model II was developed. This divided the gut
wall based on the cellular localization of enzymes into cytosolic and
membrane (endoplasmic reticulum)-bound compartments. Conceivably,
phenol entering the gut epithelium would have immediate access to the
cytosolic PSTs but would have to diffuse across the lipophilic ER
membrane to access the UGTs. The sulfate formed could either
recirculate unchanged or metabolically recycle to the parent phenol
(Tan and Pang, 2001). Metabolic ("futile") recycling of the sulfate
could explain the plateau observed in its concentration-time profile.
Phenol's access to the UGTs in the ER lumen was modeled as a
diffusional process. By setting the diffusional clearance of the
glucuronide to be smaller than its formation clearance, a diffusional
barrier to the glucuronide was modeled. The simulated reservoir
glucuronide profile (Fig. 9) now more closely resembled the
experimental observations. Interestingly, this resulted in a
significant accumulation of the glucuronide within the ER lumen. This
could explain the continued appearance of the glucuronide despite the
declining phenol concentration.
A putative rate-limiting step in intestinal glucuronidation release was
also suggested by a study with salicylamide (Barr and Riegelman, 1970).
Salicylamide was administered in vitro to rabbit everted gut sacs and
by in vivo perfusion of an intestinal loop with collection of
mesenteric vein effluent. In the in vitro experiments, whereas the
salicylamide dose was increased severalfold, the amount of glucuronide
formed remained constant. Furthermore, in the in vivo experiments,
there was an appearance of the glucuronide even though the salicylamide
concentrations were declining. With the recognition that a member of
the multidrug resistance-associated protein family is expressed in the
intestine (Peng et al., 1999; Mottino et al., 2000), the
capacity-limited efflux of the glucuronide seems reasonable.
The striking "route" dependence in the major metabolic pathway is
intriguing. Conceivably, phenol readily encountered the cytosolic PSTs
but required time to diffuse across the lipid-rich barrier of the ER to
access the active site of the UGTs. The phenyl glucuronide, being more
polar and much larger than its aglycone, would experience an even
greater diffusional barrier to its efflux from the ER lumen. Therefore,
sulfation would be expected to be the primary metabolic pathway, as
seen after vascular administration. In sharp contrast, luminal
administration of phenol yielded the glucuronide as the primary
metabolite, in addition to a much greater overall extraction. This
could be indicative of greater and perhaps more prolonged access of the
perorally administered phenol to the apically placed UGTs. In other
words, in the case of luminal dosing, the effect of cellular
localization of the UGTs was offset by their tissue localization. The
route dependence of the metabolic profile raises the possibility that
xenobiotic metabolism (or carcinogen bioactivation) might be influenced
by the route of exposure.
In conclusion, in light of the clear influence of enzyme localization
at the cellular and tissue level on intestinal clearance, the division
of the intestine into the lumen, mucosa, and serosa might in itself not
be adequate to model intestinal clearance. The mucosal compartment
could be further divided into a compartment for membrane-bound enzymes
like the UGTs and CYPs and another for cytosolic enzymes like the
sulfotransferases. Thus, models of the intestine that are
contingent on a diffusion rate-limited rather than perfusion
rate-limited distribution of compounds need further investigation.
We acknowledge the financial support of the International
Student Work Opportunity Program at the University of Minnesota. The
work described in this article was carried out in partial fulfillment
of the requirements for a Ph.D. at the University of Minnesota
(P.A.K.).
Abbreviations used are:
UGTs, uridine
diphosphoglucuronosyltransferases;
IPI, in situ perfused intestine;
SMA, superior mesenteric artery;
PST, phenol sulfotransferase;
CL, clearance;
HPLC, high-performance liquid chromatography;
ER, endoplasmic reticulum;
AUC, area under the curve;
6AC, 6-aminocarbovir.
Integrating eqs. A-1, A-2, and A-12 with respect to time from 0 to ,
eqs. A-13, A-14, and A-15 are obtained to describe mass transfer in the
reservoir, serosa, and mucosa, respectively:
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
![<UP>V<SUB>gw</SUB></UP>[<UP>C<SUB>pv,t</SUB></UP>−<UP>C<SUB>pv,0</SUB></UP>]=[<UP>X<SUB>t</SUB></UP>−<UP>X<SUB>0</SUB></UP>]+<UP>Q<SUB>res</SUB>AUC<SUB>res,t</SUB></UP>−<UP>CL<SUB>gw</SUB>AUC<SUB>pv,t</SUB></UP>−<UP>Q<SUB>res</SUB> AUC<SUB>pv,t</SUB></UP>](/content/vol30/issue5/fulltext/586/img008.gif)
-D-glucuronides formed intraluminally in rat small intestine mucosa and absorbed into the colon.
Life Sci
65:
1579-1588[CrossRef][Medline].
),
phenyl glucuronide (
), and phenyl sulfate (
) after the low
vascular dose of phenol from the reservoir and the portal samples.
) ml/min; mean (n = 2) at flow rates of 10 (
