The Relative Importance of the Small Intestine and the Liver in Phase II Metabolic Transformations and Elimination of p-Nitrophenol Administered in Different Doses in the Rat

Intestinal and hepatic function have been investigated in phase II metabolic reactions and elimination of p-nitrophenol (PNP) in the rat. A jejunal loop was cannulated and recirculated with isotonic solutions containing PNP in different concentrations (0, 20, 100, 500, 1000 μM). Samples were obtained from the perfusate at given intervals. To investigate the metabolic and excretory functions of the liver, the bile duct was cannulated, and the bile was collected. Metabolites of PNP were determined by validated HPLC (high pressure liquid chromatography) methods. The results demonstrated the relative importance of the small intestine and the liver in phase II metabolic transformations and elimination of PNP. There were significant differences between the luminal and biliary appearances of p-nitrophenol-glucuronide (PNP-G) and p-nitrophenol–sulfate (PNP-S). The PNP-G appeared in the intestinal lumen at the lower PNP concentrations (20 μM and 100 μM) at higher rate than in the bile. No significant difference was found between the intestinal and the biliary excretion of PNP-G when PNP was administered at a concentration of 500 μM. However, a reverse ratio of these parameters was observed at the administration of 1000 μM PNP. The results indicated that both the small intestine and the liver might play an important role in phase II metabolic reactions and elimination of PNP. However, the relative importance of the small intestine and the liver can be dependent on the dose of drugs.


Introduction
After administration, drug molecules must reach the site of action to be effective. Per os delivered drugs during the pharmacokinetic processes, for example, can be inactivated by enzymes in the gut wall cells and hepatocytes [1][2][3][4]. It is well known that the liver has a vital role in the biotransformation of xenobiotics. However, some new experimental results demonstrated that the extrahepatic drug metabolism could also be significant [4][5][6][7][8]. A particular aspect of intestinal metabolism is its location at the site of the entry of xenobiotics. The metabolites of exogenous compounds formed in the small intestine can be excreted by enterocytes back into the intestinal lumen and, after that, can be discharged with the feces [9,10]. Enterocytes express several metabolic enzymes that are also found in the liver, including, e.g., UDP-glucuronyltransferases, sulfotransferases, esterases, and cytochromes P450 [11][12][13][14][15][16][17][18].

Animals and Experimental Procedure
The experiments were performed according to the protocol used before [19][20][21][22][23]29,30]. Male Wistar rats (weighing 220-250 g) were used. The animals were fasted 18-20 h prior to the experiments; water was provided ad libitum. The animals were anesthetized with urethane (1.2 g/kg, i.p.). The abdomen was opened by a mid-line incision, and a jejunal loop (distance from the duodeno-jejunal flexure of about 15 cm; length of the jejunal loop of about 10 cm) was "in vivo" isolated and cannulated at its proximal and distal ends. Body temperature was maintained at 37 • C using a heat lamp. The lumen of the jejunal loop was gently flushed with warmed isotonic solution to remove digesta and food residues and then blown empty with 4-5 mL air. Perfusion through the lumen of the jejunal loop with isotonic medium containing PNP in different concentrations (0 µM, 20 µM, 100 µM, 500 µM, and 1000 µM) was carried out at a rate of 13 mL/min in a recirculation mode for 90 min. The volume of the samples obtained from the perfusion medium flowing out of the jejunal loop was 250 µL; the initial volume of the perfusate was 15 mL. The temperature of the perfusate was maintained at 37 • C. For investigation of the biliary excretion rate, the bile duct was cannulated with a PE-10 tubing and the bile collected in 15 min periods. Bile flow was measured gravimetrically, assuming a specific gravity of 1.0. Biliary excretion was calculated as the product of the concentration in bile and bile flow.
Each experimental group had eight animals. The collected samples were refrigerated (−20 • C) until analysis.
UV-Vis measurements were performed on a Pye Unicam PU 8800 UV-Vis spectrophotometer (Philips, Cambridge, UK) at ambient temperature. A Mettler Toledo MP 220 pH meter and a Mettler Toledo Inlab 413 electrode (Mettler Toledo, Budapest, Hungary) were used to adjusting the pH of the electrolyte solutions.

Analytical Conditions
The small intestine perfusate samples were analyzed by a UV-Vis RP-HPLC method developed and published earlier [29]. Briefly, the mobile phase consisted of methanol and distilled water (50:50 v/v%) containing 0.01 M TBAB. The perfusates were vortexed and centrifuged at 3000× g for 10 min before the analysis. The flow rate of the eluent was 1.2 mL/min.
The bile samples were analyzed by an RP-HPLC method with UV-Vis detection as was developed and published earlier [30]. Briefly, 50 µL of bile sample were mixed and vortexed with cold methanol in a total volume of 250 mL containing 2.5 mM ETP as an internal standard. A mixture of 0.01 M citrate buffer (pH 6.2) and methanol (47:53 v/v%) containing 0.03 M tetrabutylammonium bromide was used as a mobile phase. The flow rate of the eluent was 1.0 mL.min −1 .
In both analytical methods, the volume of the samples was 20 µL, and the detection was effected at 290 nm because this wavelength proved to be optimal for the simultaneous measurement of PNP, PNP-G, and PNP-S. Before the analysis, the temperatures of samples were allowed to rise to ambient temperature.

Calculations, Statistical Analysis
The luminal appearance and the biliary excretion of PNP metabolites (PNP-G, PNP-S) were calculated on the basis of their concentrations in the perfusion medium and the bile, and the corresponding volumes of the perfusion solution and the bile, respectively. At mass-volume conversion of the bile samples, 1 g/cm 3 was considered [36]. The data show the average values ± S.D. of eight experiments. Significant differences were calculated by a two-sample Student's t-test.

Ethical Approval
All procedures were carried out on animals according to the Hungarian Animal Protection Act (Hungarian Act XXVIII, 1998). The study was approved by the Ethics Committee on Animal Research of the University of Pécs.

Results
The luminal disappearance of PNP (500 µM) from the intestinal perfusion solution is shown in Figure 1.
The luminal appearance and the biliary excretion of PNP metabolites (PNP-G, PNP-S) were calculated on the basis of their concentrations in the perfusion medium and the bile, and the corresponding volumes of the perfusion solution and the bile, respectively. At mass-volume conversion of the bile samples, 1 g/cm 3 was considered [36]. The data show the average values ± S.D. of eight experiments. Significant differences were calculated by a two-sample Student's t-test.

Ethical Approval
All procedures were carried out on animals according to the Hungarian Animal Protection Act (Hungarian Act XXVIII, 1998). The study was approved by the Ethics Committee on Animal Research of the University of Pécs.

Results
The luminal disappearance of PNP (500 µM) from the intestinal perfusion solution is shown in Figure 1.

Luminal Appearance of p-Nitrophenol-Glucuronide (PNP-G) and p-Nitrophenol-Sulfate (PNP-S) in the Small Intestine (Proximal Jejunum) Measured in 90 min Perfusion of 500 µM PNP
During the perfusion of the 500 µM PNP solution, the PNP metabolites (PNP-G, PNP-S) appeared in the intestinal lumen. The appearance of PNP-G increased linearly in 90 min of the perfusion period. The PNP-S-as expected-appeared in the intestinal lumen in a much lower amount. The result is in agreement with the known biochemical difference (low affinity-high capacity vs. high affinity-low capacity, respectively) of the two competing phase II metabolic pathways. (Figure 2).  The PNP-S-as expected-appeared in the intestinal lumen in a much lower amount. The result is in agreement with the known biochemical difference (low affinity-high capacity vs. high affinity-low capacity, respectively) of the two competing phase II metabolic pathways. (Figure 2).

Biliary Excretion of PNP-G and PNP-S Measured in 90 min Perfusion of 500 µM PNP in the Small Intestine (Proximal Jejunum)
Under the same experimental conditions (intestinal perfusion of 500 µM PNP solution), the PNP-G and PNP-S metabolites were excreted into the bile as well ( Figure 3). The biliary excretion rate of PNP-G, similar to its intestinal appearance, was significantly higher than that of the PNP-S. The course of the respective curves in the intestinal perfusate and the bile is similar. It is a remarkable finding, at this perfusate concentration, that the amount of the excreted PNP-S is much higher in the bile than in the intestinal perfusate. On the other hand, the amounts of PNP-G in the two fluids are similar (see also Figures 5 and 6). Under the same experimental conditions (intestinal perfusion of 500 µM PNP solution), the PNP-G and PNP-S metabolites were excreted into the bile as well ( Figure 3). The biliary excretion rate of PNP-G, similar to its intestinal appearance, was significantly higher than that of the PNP-S. The course of the respective curves in the intestinal perfusate and the bile is similar. It is a remarkable finding, at this perfusate concentration, that the amount of the excreted PNP-S is much higher in the bile than in the intestinal perfusate. On the other hand, the amounts of PNP-G in the two fluids are similar (see also Figures 5 and 6). Sci. Pharm. 2020, 88, x FOR PEER REVIEW 6 of 14

Biliary Flow Measured in Control and PNP-Perfused Rats Measured in 90 min Perfusion of 500 µM PNP in the Small Intestine (Proximal Jejunum)
The biliary flow rates in control and PNP-perfused rats measured in a 90 min perfusion of 500 µM PNP in the small intestine are demonstrated in Figure 4. The bile production was relatively constant (0.23 mL/15 min period) in the control rats and was not changed significantly during the biliary excretion of PNP-G and PNP-S. These results show that the luminal perfusion of PNP did not influence the bile production of the liver.
The cumulative luminal appearance and the biliary excretion rate of PNP-G and PNP-S are demonstrated in Figures 5 and 6, respectively. Data refer to the amounts of PNP-G and PNP-S measured over 90 min of luminal perfusion of PNP in different concentrations (20 µM, 100 µM, 500 µM, and 1000 µM) of PNP.   Figure 5 illustrates the cumulative luminal appearance and biliary excretion of PNP-G in the same figure and at the same scale, allowing for an adequate comparison of the metabolic and excretory function of the small intestine and the liver. As can be seen, at the lower PNP concentrations (20 µM and 100 µM), the luminal appearance of PNP-G exceeded the biliary excretion of this metabolite; however, at the 500 µM PNP concentration, no significant difference was found between these values. Moreover, when the PNP concentration was further elevated (up to 1000 µM), the biliary excretion of PNP-G was significantly higher than the luminal appearance of the PNP-glucuronide. These results suggest the saturability of the luminal appearance of PNP-G; however, the biliary excretion showed a continuous and dose-dependent increase while the perfused PNP concentration was elevated up to 1000 µM.  Figure 6 demonstrates the cumulative luminal appearance and the biliary excretion of PNP-S under the same experimental conditions as was mentioned with data of Figure 5.      function of the small intestine and the liver. As can be seen, at the lower PNP concentrations (20 µM and 100 µM), the luminal appearance of PNP-G exceeded the biliary excretion of this metabolite; however, at the 500 µM PNP concentration, no significant difference was found between these values. Moreover, when the PNP concentration was further elevated (up to 1000 µM), the biliary excretion of PNP-G was significantly higher than the luminal appearance of the PNP-glucuronide. These results suggest the saturability of the luminal appearance of PNP-G; however, the biliary excretion showed a continuous and dose-dependent increase while the perfused PNP concentration was elevated up to 1000 µM.   Figure 5.

Cumulative Luminal Appearance and Biliary Excretion of PNP-S Measured in 90 min
It can be seen that PNP-S appeared in a considerably lower amount in the intestinal lumen than in the bile. In contrast to the dose-dependent increase in the biliary excretion of the sulfate metabolite (PNP-S), the luminal appearance of PNP-S remained at a relatively low level even at higher PNP concentrations. The results are in agreement with the previously demonstrated quantitative difference between the differences between the intestinal and biliary excretions of PNP-S, which were statistically significant at each PNP concentration.

Discussion
p-Nitrophenol has been reported to be effectively absorbed from the small intestine [20][21][22]29]. To obtain comparable data on the intestinal absorption as well as intestinal and hepatic metabolism from the same animals, HPLC-UV-Vis determination of the disappearance of PNP from the small intestine (proximal jejunum) was carried out. As shown in Figures 1 and 2, the luminal amount of PNP decreased rapidly and continuously, and PNP-G and PNP-S metabolites appeared in the lumen of the in vivo isolated jejunal loop. At the same time, both PNP-G and PNP-S were also excreted into the bile (Figure 3).
The appearance of the glucuronide metabolite in the perfusate is in accordance with the results of our previous investigation with capsaicinoids [37]. On the contrary, results with ibuprofen-an NSAID with carboxyl moiety-showed no respective glucuronide in the perfusate [38]. The explanation of these differences can be rationalized by previous results, which demonstrated that the conjugation reaction on the carboxyl moiety with glucuronic acid is mediated by UGT2B1 (UDP-glucuronosyltransferase 2B1), in the rat [39]-UGT2B7 in humans [40]-which is expressed at an insignificant level in the rat small intestine. On the contrary, formation of the phenol-glucuronide conjugates is presumably associated with the preference of the UGT1A1, UGT1A6, and UGT1A8 isoforms, which catalyze ether-O-glucuronidation reactions [41,42].
Luminal appearance and biliary excretion of the PNP-G and PNP-S metabolites were investigated with experiments with different PNP concentrations (20 µM, 100 µM, 500 µM, and 1000 µM) in the small intestinal perfusate. These experiments demonstrated dose-dependent changes in the relative luminal appearance of PNP-G. At the lower PNP concentrations (20 µM and 100 µM), the luminal appearance of PNP-G was higher than its biliary excretion. However, while elevating the substrate (PNP) supply, the luminal appearance of PNP-G tended to be saturated: at a 500 µM PNP concentration, there was no significant difference between the intestinal and biliary excretion of PNP-G. Furthermore, at the 1000 µM PNP concentration, the biliary excretion exceeded the luminal appearance of PNP-G. Theoretically, excretion of the PNP-G (and the PNP-S) metabolites formed in the small intestine to the bile cannot be excluded. In our earlier study, it was demonstrated that the activity of UDP-glucuronyltransferase and sulfotransferase (SULT) was about three times higher in the rat liver than in the small intestine. The activity of the β-glucuronidase was about six times higher and that of the of the arylsulfatase, which was approximately seven times greater in the liver than in the small intestine [21]. Taking into consideration these experimental data, it can be concluded that most of the PNP-G and the PNP-S conjugates excreted to the bile are formed in the liver. Further experiments are needed to obtain a more detailed quantitative aspect of the question.
The luminal appearance of PNP-S was lower than the biliary excretion of the sulfate conjugate and showed a tendency to saturation. However, the biliary excretion of the sulfate metabolite was increased with elevation of the PNP concentration of the perfusate. Similar to our results, different rates of sulfation were measured by other authors in the human liver and intestinal cytosol with various drugs [2].
These results demonstrate that in contrast to the saturability of the metabolic and excretory function of the small intestine, the hepatic elimination of PNP-glucuronide is continuously elevated with the increasing concentrations (20-1000 µM) of the luminally perfused PNP. Similar results were also found by other authors while investigating glucuronidation of drugs by the human liver and intestinal microsomes [12,13,43].
Activity and expression of the relevant metabolic enzymes (glucuronyl transferases/glucuronidases and sulfotransferases/sulfatases) and transporters are different in the two organs. The main UGT isoforms expressed in the rat liver and intestine are UGT1A1, UGT1A6, UGT1A7, and UGT1A8 [44,45]. In rats, the PNP-dependent UGT activity of the liver has been reported to be about ten times higher than that of the different segments of the small intestine [46]. In rodents, cytosolic sulfotransferases are present in the liver, gut, adrenal, kidney, lung, skin, brain, and other extrahepatic tissues. The overall expression level of SULTs in the intestine, however, is much lower than that in the liver [47]. In published reports, many glucuronide conjugates were demonstrated to be substrates of ATP-dependent efflux (ABC) transporters, including breast cancer resistance protein (BCRP) and multidrug resistance-associated protein (MRP) 1/2/3/4 [48][49][50][51]. These efflux transporters function as efflux pumps to extrude intracellular conjugates and facilitate their excretion into the lumen and bile or uptake into the blood. BCRP and MRP2, which can transport glucuronide conjugates into the lumen and the bile, are rather non-selective transporters and accept most of the glucuronides. The expression of MRP2 is higher in the liver than along the intestine. [52,53]. Besides glucuronide conjugates, BCRP can also transport sulfate conjugates. The kinetics of the metabolic enzymes and the transport of most of the substrates follows the Michaelis-Menten kinetics [54]. Accordingly, the observed differences could be the result-at least partially-of the different local concentrations of the substrate (PNP) in the enterocytes and the hepatocytes.
Another aspect of the concentration-dependent differences in the metabolic and excretory function of the small intestine and the liver is the microsomal uncoupling effect of PNP [55]. Both conjugation reactions, as well as the transportation of the metabolites, are ATP-dependent processes. Since the local concentration of PNP is higher in the epithelial cells of the small intestine than in the liver, the higher PNP concentrations (500 µM and 1000 µM) have a higher impact on the ATP-dependent metabolic transformations and transportations in the small intestine. Since glucuronidation has a much higher capacity than sulfation, the changes are more highly expressed in the glucuronidation processes.
These results indicate that both the small intestine and the liver can conjugate PNP with glucuronic acid and sulfate. Our data show that at the lower doses (20 µM and 100 µM), the small intestine can rapidly and efficiently metabolize PNP to form PNP-G and PNP-S. At these concentrations, the luminal appearance of PNP-G was higher than its biliary excretion; i.e., at the 20 µM and 100 µM PNP concentrations, the metabolic activity of the small intestine is more important than that of the liver. In our experimental protocol, the data of the luminal appearance of the conjugates represent only the function of the cannulated jejunal loop (length about 10 cm). In contrast, the biliary excretion rate indicates the metabolic function of the whole liver. On the other hand, when the concentration of PNP was elevated (500 µM and 1000 µM), and the luminal appearance of PNP-G tended to be saturated, the relative metabolic activity of the liver became more pronounced, and the biliary excretion of PNP-G exceeded its luminal appearance.
Formation of phenyl glucuronides and phenyl sulfates are generally considered to be detoxification processes [56]. As our present and previous results [22,23,29,30,34] demonstrate, the formed phenol conjugates can be reexcreted into the gastrointestinal system both by the liver and the small intestine. At the lower PNP concentrations (up to 500 µM), a higher fraction of the absorbed dose is reexcreted into the small intestine in the PNP-G form. Accordingly, reduction of the phenolic drug's dose results in its lower bioavailability. Furthermore, the glucuronide and the sulfate conjugates of phenolic drugs can be hydrolyzed by gut microbial glucuronidases and sulfatases. Acetylases, methylases, and glucuronidases are particularly widespread in the gut microbiome, as they are a rich carbon source for energy metabolism. Accordingly, the phenolic drugs with high efficacy and potency-after reactivation of the gut microbiome-can exert their (desired or undesired) effects on the lower part (colon) of the gastrointestinal system [57].

Conclusions
Our investigations showed-although the liver has the highest metabolic capacity-the intestinal metabolism to have significance in the case of substrates (drugs) that can be glucuronidated and sulfated. Such metabolic transformations have higher impact on the biotransformation of drugs applied in low doses. Under such conditions, the formed phenolic metabolites can be effectively excreted back to the small intestine, resulting in a low bioavailability of the pharmacons. Using the same drug in somewhat higher concentrations, however, the metabolic capacity of the enterocytes can be saturated, allowing a higher portion of the dose to reach the liver. The enzyme activities, the types and localizations of transporters, and the integrity of the ATP required processes are also able to influence the metabolized and excreted amounts of substrates. These data draw attention to the importance of the metabolic activity of the small intestine whose effect can result in different pharmacokinetic and pharmacodynamic features of the phenolic substrates used in different doses.