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Over the past decade the important role of the gastrointestinal (GI) tract and especially the small intestine in the presystemic metabolism of orally administered drugs has become apparent [1–3]. It has been shown that the metabolism of some orally administered drugs is unaffected in patients with impaired liver function . Kolars et al. showed that metabolites of cyclosporine were detected in portal venous blood during the anhepatic phase of liver transplantation when the drug was administered directly to the small bowel. Furthermore, Paine et al. have shown that midazolam is metabolized in the gut wall.
The cytochromes P450 (CYP) enzymes belong to a superfamily of membrane-bound haeme-containing proteins responsible for the metabolism of several endogenous compounds such as steroids and fatty acids, as well as drugs and other xenobiotics [7, 8]. Three CYP gene families are predominantly involved in drug metabolism, namely CYP1, CYP2 and CYP3, many members of which are induced or inhibited by a range of compounds .
Another determinant of the oral bioavailability of drugs is P-glycoprotein (P-gp) which is encoded for by the multidrug resistance gene (MDR1). P-gp is a membrane-bound transporter protein, which was first identified as being responsible for the development of multidrug resistance . P-gp acts as a cellular efflux pump in an ATP-dependent process . P-gp is present in mucosal cells of the GI tract, in the biliary canaliculi of the liver, and in the brush border of the proximal tubules of the kidney, where it contributes to the excretion of substances into the gut, bile and urine, respectively . Schwab et al. suggested that one of the polymorphisms of the MDR1 gene, C3435T, which leads to decreased P-gp expression, is associated with increased susceptibility to ulcerative colitis. Furthermore, it has been shown that mdr1a knockout mice develop a form of this disease, which can be prevented by antibiotics . These findings suggest that P-gp plays a protective role against intestinal bacteria and toxins.
The use of molecular techniques and especially the real-time reverse transcription-polymerase chain reaction (RT-PCR) makes it possible to detect and quantify RNA in very small samples .
The aim of this study was to study the expression of CYP2E1, CYP3A4, CYP3A5 and MDR1 mRNA along the GI tract within the same individual.
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The percentage of biopsies with measurable amounts of each mRNA is shown in Figure 1. CYP2E1 mRNA expression was detected in 81–96% of the samples at the different sites along the GI tract. CYP3A5 mRNA was detected in all samples at all locations, whereas CYP3A4 mRNA detection varied between 41% and 96% and P-gp mRNA between 59% and 96%. However, the data from all samples, and also those with a Ct value above 38, are included in Figure 2, since they all expressed measurable concentrations of b-actin-specific mRNA, that is, a Ct value below 38. Because of the small size of the forceps used, biopsies contained only mucosal tissue. The majority of the patients were diagnosed with irritable bowel disease (Table 1) and as expected there was no inflammation in these biopsies. When all samples were considered, no difference in the median b-actin mRNA expression was observed in those that showed inflammation compared with those that did not.
Figure 2. Box plots of the CYP-specific/b-actin mRNA ratio for each enzyme at different locations of the human gastrointestinal tract (data from all samples included). Arrows indicate the location with the highest mRNA expression for each enzyme. Median (□), 25%–75% ( ), Min-Max ( )
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The expression of CYP2E1 mRNA was highest at all sites (P < 0.05 to P < 0.0001) except in right colon, where it was equal to the expression of CYP3A5 mRNA (Figure 2 and Table 2). The highest expression of CYP2E1 mRNA was observed in the stomach and duodenum (P < 0.01), but there was a substantial interindividual variation (Kendall coefficient of concordance = 0.16). In the duodenum the expression of CYP2E1 mRNA exceeded that of CYP3A4, CYP3A5 and P-gp by approximately seven-, six- and 300-fold, respectively.
Table 2. Median (25-75% quartile) mRNA/â-actin ratios in different parts of the gastrointestinal tract
|Stomach||0.061 (0.017-0.29)||0.000053 (0.0000040-0.0036)||0.0058 (0.0026-0.020)||0.00002 (0.0000060-0.00034)|
|Duodenum||0.053 (0.0078-0.33)||0.0072 (0.00026-0.036)||0.0095 (0.0024-0.019)||0.00019 (0.000014-0.0014)|
|Right colon||0.017 (0.00010-0.06)||0.00006 (0.0000030-0.0023)||0.0028 (0.0012-0.0048)||0.00042 (0.00011-0.0022)|
|Left colon||0.023 (0.0022-0.13)||0.0001 (0.0000040-0.00095)||0.0029 (0.0011-0.0074)||0.0015 (0.00030-0.0036)|
|Rectum||0.012 (0.0032-0.034)||0.000032 (0.0000060-0.00031)||0.0018 (0.00076-0.0030)||0.00073 (0.00011-0.0026)|
The expression of CYP3A4 mRNA was highest in the duodenum (P < 0.001), with no or low expression in the stomach and colon (Figure 2 and Table 2), and showed great interindividual variability (Kendall coefficient of concordance = 0.25).
The expression of CYP3A5 mRNA was highest in the duodenum and stomach (P < 0.000 001) and was less in the colon (Kendall coefficient of concordance = 0.34) (Figure 2 and Table 2).
To validate our finding that the expression of CYP3A5 mRNA was generally higher than that of CYP3A4, we analysed expression of these CYP genes in four different liver samples and one additional intestinal sample, obtained at surgery. In addition, another laboratory analysed the same livers and intestine using a similar method and found comparable results.
For P-gp, mRNA expression increased from stomach and duodenum to left colon (P < 0.000 001] a Kendall coefficient of concordance = 0.34) (Figure 2 and Table 2).
The interindividual variability in expression at each site in the GI tract was again very high. Each individual showed a similar expression pattern for the investigated CYP enzymes and P-gp throughout the GI tract.
We did not find any difference in mRNA expression between smokers (n = 8) and nonsmokers (n = 19), or between those who had an alcohol intake of less than 50 g compared with those who consumed more than that (n = 7) during the week before investigation, for any of the CYPs studied. Furthermore, there were no sex differences in expression (F = 15, M = 12), or differences between patients on regular medication (n = 16) compared with patients not taking medication (n = 11).
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To our knowledge, this is the first study to characterize the expression of CYP enzymes and P-gp along the length of the GI tract of the same individual in a large patient population. We determined the mRNA expression of three important CYP enzymes and the MDR1 gene at five different sites in 27 patients. The methods used were sensitive enough for several analyses of the very small amount of biopsy material (about 5 mg wet weight) that can be obtained during the endoscopic investigation. However, there was insufficient to study these CYPs or P-gp at a functional or protein level.
We found that CYP2E1 had the highest mRNA expression of the CYPs studied in all parts of the GI tract. Our findings are consistent with those of other investigators who have demonstrated the presence of CYP2E1 in the terminal ileum and colon of patients with irritable bowel disease, Crohn's disease and ulcerative colitis, respectively . However, because of post-transcriptional regulation , different authors have failed to find a relationship between CYP2E1 mRNA and protein concentration [21, 22]. This suggests that there is no clear association between CYP2E1 mRNA expression and metabolic activity.
It has been claimed that there is a relationship between mRNA and protein expression for CYP3A4 and CYP1A2 [21, 22], although the data are conflicting, and our group has not been able to confirm this assertion (unpublished data).
Watkins and coworkers  have published data on the relative abundance of CYP3A4 in the intestinal mucosa, and protein expression and catalytic activity have been reported to decrease longitudinally along the small intestine . We found higher CYP3A4 mRNA expression in the duodenum than in the stomach and the colon, which is in agreement with earlier reports based on human transplant donors [25, 26].
It has been shown that CYP3A4 in the gut is an important determinant of the bioavailability of cyclosporine, because approximately one-half to two-thirds of its metabolism occurs presystemically when the drug is orally administered [1, 27]. Furthermore, other drugs such as midazolam are metabolized to a varying degree by CYP enzymes in the gut . It has also been shown that plasma concentrations of digoxin (with which two of the patients were treated) decrease with concomitant administration of rifampicin. This may be explained by an increase in intestinal P-gp during treatment with this inducing agent .
CYP3A5 mRNA expression was found in all the samples studied. Earlier studies [29, 30] demonstrated the presence of CYP3A5 mRNA in the small intestine, but CYP3A5 protein was not detected. In contrast, Lown et al. found CYP3A5 protein in the small bowel of 70% of the patients studied, and they suggested that this enzyme may be expressed more in the intestine than in the liver. Kolars et al. detected CYP3A5 and CYP3A4 mRNA in all regions of the GI tract using microsomes from a human organ donor.
The median concentration of CYP3A5 mRNA was highest in duodenal mucosa but the difference was not as pronounced as for CYP3A4 mRNA. Although CYP3A4 is considered to be the major CYP enzyme in the small intestine . Lown et al. described similar concentrations of CYP3A4 and CYP3A5 protein in enterocytes. In the present study we found similar expression of CYP3A4 and CYP3A5 mRNA in the duodenum. The expression of CYP3A5 protein is polymorphic, and individuals with at least one CYP3A5*1 allele express greater amounts of the enzyme. Based on our data, the expression of CYP3A5 mRNA is monomorphic, since the CYP3A5*3 allelic variant also expresses mRNA to a varying degree .
In the colon there was greater expression of CYP3A5 mRNA compared with that of CYP3A4, which is in agreement with findings of McKinnon et al.. Other data also indicate that CYP3A5 is the predominant CYP3A enzyme in colonic mucosa [26, 32]. Furthermore, there appears to be greater expression of CYP3A5 in the stomach than CYP3A4 , which is consistent with the findings of the present study.
It seems that there is a relatively low CYP3A phenotypic variability among healthy adults compared with that seen in archived intestinal and liver tissue samples . One possible explanation for this is the different rates of CYP3A4 and CYP3A5 degradation during the harvesting and cold storage of the tissues , which could explain the discrepancies between the relative proportion of CYP3A4 and CYP3A5 mRNA expression in our study and previous reports [29, 30]. It is unclear whether the rates of degradation of CYP3A4 and CYP3A5 are different in the GI tract compared with the liver.
Lown et al. found that variation in intestinal P-gp accounted for about 30% of the variability in peak blood concentration of orally administered cyclosporine. We found a continuous increase in MDR1 expression along the GI tract to distal descending colon. Our findings are in agreement with those of Fojo et al., although this report was based on colon samples from different patients and only one jejunum and rectum sample. Mouly and Paine  showed an increase in P-gp expression from proximal to distal small intestine. It has been suggested by Stephens et al. that the ileum and distal colon are regions with high permeability for xenobiotics and that this is compensated for by enhanced expression of P-gp. The distribution pattern of MDR1 mRNA is opposite to that of CYP3A4 mRNA. These two proteins have overlapping substrate specificity and tissue distribution . High expression of P-gp in the more distal part of the GI tract may be important in expelling xenobiotics not yet metabolized by CYP3A.
We found no influence of smoking, alcohol intake or sex on mRNA concentrations. However, the groups were quite small, and hence these findings must be interpreted with caution. There was no detectable relationship between the medication taken by patients and mRNA expression. However, the reported drug consumption was very heterogeneous among the patients.
We conclude that among the CYP enzymes investigated CYP2E1 mRNA expression was highest throughout the GI tract, with that of CYP3A being greatest in the duodenum. This finding is relevant because of the emerging role of the gut in the metabolism of a large number of clinically important drugs. MDR1 mRNA expression increased continuously from duodenum to colon, a finding that may be linked to a natural role for P-gp in the protection against xenobiotics produced by the intestinal microflora.