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OXIDATIVE METABOLISM OF THE ALKALOID RUTAECARPINE BY HUMAN CYTOCHROME P450

National Research Institute of Chinese Medicine (Y.-F.U., M.-J.D., S.-Y.W., C.-F.C.); Department of Pharmacology, National Yang-Ming University (Y.-F.U., W.-C.J., L.-K.H., C.-F.C.); Graduate Institute of Medical Sciences, Taipei Medical University (Y.-F.U.); and Mackey Medicine, Nursing and Management College (W.-C.J.), Taipei, Taiwan, R.O.C.; and Qing-Dao University, Qing-Dao, China (C.-F.C.)

(Received October 13, 2005; accepted February 13, 2006)

	Abstract

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Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by -naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V_max values of 1388 to 1893 pmol/min/nmol P450, K_m values of 4.1 to 9.5 µM, and K_i values of 45 to 103 µM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC₅₀ values of 2.56 ± 0.04, 2.57 ± 0.11, and 0.09 ± 0.01 µM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.

In human liver, CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 are the main P450 forms responsible for drug oxidation (Guengerich, 1995). CYP1A2, CYP2C, CYP2D6, CYP2E1, and CYP3A constitute approximately 13%, 18%, 2%, 7%, and 29% of the total P450 content, respectively (Shimada et al., 1994). CYP2C9 represents about half to 75% of total CYP2C (Lasker et al., 1998). CYP3A4 is the most abundant P450 form in human liver samples. CYP1A1 and CYP1B1 are expressed in low levels in human liver, but are inducible in the liver and play important roles in the activation of protoxins in induced liver and extrahepatic tissues. CYP2B6 constitutes approximately 0.2% of the total hepatic P450 content. Although the content of CYP2B6 was low in human liver, CYP2B was studied because of its main role in rat rutaecarpine hydroxylation (Lee et al., 2004). -Naphthoflavone (-NF), orphenadrine, sulfaphenazole, quinidine, 4-methylpyrazole, and ketoconazole preferentially inhibited CYP1A2, CYP2B6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 activities, respectively (Chang et al., 1994; Balawin et al., 1995; Bourrie et al., 1996). Thus, to identify the P450 forms participating in human rutaecarpine hydroxylations, the effects of P450 inhibitors were analyzed using human liver microsomes, and rutaecarpine hydroxylation activities were determined using Escherichia coli membranes expressing human P450s. Among expressed P450 forms, our results showed that CYP1A1 had the highest activity. The kinetic parameters of CYP1-catalyzed hydroxylations were determined.

View larger version (38K): [in this window] [in a new window]   FIG. 1. A, the HPLC chromatograms of rutaecarpine hydroxylation metabolites by recombinant human CYP1A1 (A) and human liver microsomes (B). Metabolites, 10-, 11-, 12-, and 3-hydroxyrutaecarpine appeared at 16, 20, 31, and 35 min, respectively. Microsomes were prepared and activities were determined as described under Materials and Methods. B shows the chromatogram of microsomal hydroxylation metabolites by human liver samples HL1. B, microsomal rutaecarpine hydroxylation activities of human liver samples. Results represent the mean of duplicate determinations.

	Materials and Methods

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Microsomal Preparation and Bicistronic Human P450 Expression. Four Chinese liver samples (HL1–4) were obtained from patients who underwent liver resection in National Taiwan University Hospital (Taipei, Taiwan). Four liver samples (HL5–8) of white people were obtained from Dr. F. Peter Guengerich (Vanderbilt University, Nashville, TN) through Tennessee Donor Services (Nashville, TN). Microsomes of frozen human liver samples were prepared following the method of Guengerich (1994). Bicistronic human P450 plasmids were kindly provided by Dr. F. Peter Guengerich. P450 constructs consisting of the coding sequence of a P450 followed by that of NADPH-P450 reductase were transformed to E. coli DH5 by electroporation. Bacterial membrane fractions were prepared following the method of Parikh et al. (1997). Microsomes and bacterial membrane fractions were stored at –75°C until use.

Enzyme Assays. Microsomal protein concentration was determined by the method of Lowry et al. (1951). P450 content was measured by the spectrophotometric method of Omura and Sato (1964). 7-Ethoxyresorufin O-deethylation activity was determined by measuring the fluorescence of resorufin (Pohl and Fouts, 1980). Rutaecarpine hydroxylation activity was determined as described before (Ueng et al., 2005). Incubation mixtures (1 ml total volume) contained 1 mg/ml microsomal protein, a NADPH-generating system, and various concentrations of rutaecarpine as indicated under Results. The NADPH-generating system consisted of 10 mM G6P, 0.5 mM NADP, and 0.25 U/ml G6P dehydrogenase. For E. coli membranes expressing human CYP1, 20 pmol of P450 was added in the assay. For membranes expressing human CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP3A4, 100 pmol of P450 was added in the assay. Hydroxylation metabolites were separated by HPLC using a C18 column [4.6 x 250 mm, 5 µm, Agilent Technologies Inc. (Palo Alto, CA); or 5C18-MS-II, Cosmosil, Nacalai Tesque Inc. (Kyoto, Japan)] and a mobile phase containing 10% methanol and 26% acetonitrile in 0.04% formic acid at a flow rate of 1 ml/min. A UV detector, L-7420 (Hitachi Ltd., Tokyo, Japan), was used and metabolites were detected by measuring the absorbance at 344 nm. The amounts of hydroxyrutaecarpines were determined using synthetic standards. Orphenadrine and 4-methylpyrazole were dissolved in water. -NF, sulfaphenazole, quinidine, and ketoconazole were dissolved in ethanol. For the inhibition study, the concentrations of -NF, orphenadrine, sulfaphenazole, quinidine, 4-methylpyrazole, and ketoconazole in the assay were 10, 300, 1, 10, 20, and 1 µM, respectively. The final concentration of ethanol was less than 0.4%. The same concentration of ethanol was added in the control incubation.

Binding Spectral Analysis. Binding spectra were analyzed by using tandem cuvettes, which have two separate chambers in one cuvette. Various concentrations of 10-hydroxyrutaecarpine and E. coli membrane suspension containing 0.2 µM P450 in 0.1 M potassium phosphate buffer, pH 7.4, were added in both sample and reference cuvettes at room temperature. In the reference cuvette, E. coli membrane suspension and 10-hydroxyrutaecarpine in potassium phosphate buffer were kept separated in two chambers. After mixing E. coli membrane suspension with 10-hydroxyrutaecarpine in the sample cuvette, the difference spectrum was recorded from 350 nm to 450 nm. The spectral dissociation constant (K_s) and maximal absorbance change (A_max) were calculated by nonlinear regression without weight according to the equation A = I · A_max/(K_s + I), where I is the inhibitor concentration (Sigma Plot, SPSS Inc., Chicago, IL).

Data and Statistical Analyses. Substrate inhibition can be regarded as a form of uncompetitive inhibition (Houston and Kenworthy, 2000). Thus, kinetic parameters of CYP1A1-catalyzed rutaecarpine hydroxylations were calculated according to uncompetitive inhibition by substrate. Values of velocities () at various substrate concentrations, S, were fit using nonlinear least-squares regression without weight due to the equation: = V_max · S/(K_m + S(1 + S/K_i)), where V_max and K_i are the maximal velocity and inhibition constant, respectively (Sigma Plot, SPSS Inc.). The concentrations of chemicals required for 50% inhibition of 7-ethoxyresorufin O-deethylation activity (IC₅₀) were calculated by curve fitting (Grafit; Erithacus Software Ltd., Staines, UK). For inhibitory kinetic analysis, values of velocities () at various substrate concentrations, S, were fit using nonlinear least-squares regression without weight due to the equation, consistent with the competitive inhibition according to the Michaelis-Menten equation: = V_max · S/(K_m[1 + (I/K_i)] + S), where I and K_i are the inhibitor concentration and inhibition constant, respectively (Sigma Plot, SPSS Inc.). The significant relationship of pairs of variables was analyzed by Spearman rank correlation test using the software Sigma Stat 2.03 (SPSS, Inc., Chicago, IL). A p value <0.05 was considered statistically significant.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Relationship between rutaecarpine hydroxylation activity and P450 concentrations in the assay. Rutaecarpine hydroxylation activity of bacterial membrane expressing CYP1A1 (), CYP1A2 (), CYP1B1 (), CYP2C9 (), CYP2D6 (), and CYP3A4 () was determined using 50 µM rutaecarpine in the reaction mixture.

	Results

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Rutaecarpine Hydroxylation by Human P450 Forms. At rutaecarpine concentrations less than 50 µM in the assay, some P450-catalyzed rutaecarpine hydroxylations were under the detection limit. Thus, 50 µM rutaecarpine was used in the assay of all bicistronic human P450 forms determined. Hydroxylation product formation was linear at CYP1A1 and CYP1A2 concentrations in the range of 1 to 20 pmol P450/ml (Fig. 2). However, at the concentrations higher than 20 pmol P450/ml, the activities approached the plateau because of substrate saturation. For CYP2C9, CYP2D6, and CYP3A4, hydroxylation was linear at P450 concentrations up to 100 pmol P450/ml. Thus, 20 pmol P450/ml was used in the assay of CYP1 members and 100 pmol P450/ml was used in the assay of the other P450s. Rutaecarpine hydroxylation by CYP1A1 was linear with the incubation time up to 20 min (Fig. 3). Thus, the formation of hydroxylation products was determined at 20 min of incubation. HPLC analysis of rutaecarpine hydroxylation products showed that CYP1A1-catalyzed hydroxylation formed 10-, 11-, 12-, and 3-hydroxyrutaecarpine (Fig. 1). Determination of rutaecarpine hydroxylation by E. coli membrane expressing human P450 showed that CYP1A1 had the highest 10-, 11-, 12-, and 3-hydroxylation activities (Table 2). 10-Hydroxyrutaecarpine was the most abundant metabolite of rutaecarpine hydroxylation by CYP1A1. CYP1A2 had rutaecarpine 11-, 12-, and 3-hydroxylation activity. In contrast, no 10-hydroxyrutaecarpine was detected. CYP1B1 had rutaecarpine 10- and 3-hydroxylation activities. However, an additional peak appeared at 14 min, which also appeared as a metabolite by some human liver samples [Fig. 1A (B)]. The structure of this metabolite was not identified. Rutaecarpine 10-, 11-, 12-, and 3-hydroxylation activities were detected with CYP2C9 and CYP2D6. CYP2E1 showed rutaecarpine 11-hydroxylation activity and CYP3A4 showed rutaecarpine 10- and 3-hydroxylation activities. In contrast, CYP2A6 had no detectable rutaecarpine hydroxylation activity.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Relationship between CYP1A1-catalyzed rutaecarpine hydroxylation and the incubation time of the assay. Rutaecarpine hydroxylation activity of bacterial membrane expressing CYP1A1 was determined using 20 pmol of P450 and 50 µM rutaecarpine in the reaction mixture. The formation rates of 10-hydroxyrutaecarpine (), 11-hydroxyrutaecarpine (), 12-hydroxyrutaecarpine (), and 3-hydroxyrutaecarpine () were determined. Results represent the means of duplicate determinations.

Kinetic Analysis of CYP1-Catalyzed Rutaecarpine Hydroxylation. For CYP1A1-catalyzed rutaecarpine hydroxylation, the velocities decreased at rutaecarpine concentrations higher than 25 µM (Fig. 4A). At low concentrations of rutaecarpine (5 µM), the plot did not follow a hyperbolic pattern (Fig. 4B). The Lineweaver-Burk plot of CYP1A1-catalyzed rutaecarpine hydroxylation showed a nonlinear pattern (Fig. 4C). Kinetic analysis was performed according to the substrate inhibition pattern as described under Materials and Methods. The results generated kinetic parameters of CYP1A1 for each hydroxylation as follows: 10-hydroxylation, V_max = 1799 ± 326 pmol/min/nmol P450, K_m = 5.1 ± 2.0 µM, K_i = 103 ± 53 µM (r = 0.97); 11-hydroxylation, V_max = 1535 ± 292 pmol/min/nmol P450, K_m = 4.1 ± 1.8 µM, K_i = 105 ± 61 µM(r = 0.96); 12-hydroxylation, V_max = 1388 ± 350 pmol/min/nmol P450, K_m = 4.6 ± 2.6 µM, K_i = 91 ± 60 µM (r = 0.95); 3-hydroxyrutaecarpine, V_max = 1893 ± 607 pmol/min/nmol P450, K_m = 9.5 ± 5.1 µM, K_i = 45 ± 27 µM (r = 0.97). The Eadie-Hofstee plot showed a convex curve indicating positive cooperation (Fig. 4D). At a substrate concentration higher than 10 µM, the Hill plot did not show linearity (Fig. 4E). At the linear range (0.75–8 µM), results of linear regression showed cooperativities of 1.6 (r = 0.96), 1.7 (r = 0.96), 1.4 (r = 0.96), and 1.5 (r = 0.99) for 10-, 11-, 12-, and 3-hydroxylation, respectively (Fig. 4E, inset plot).

View larger version (35K): [in this window] [in a new window]   FIG. 4. Kinetic analyses of rutaecarpine hydroxylations by human CYP1A1. A, velocity () versus rutaecarpine concentration (S) plot of CYP1A1-catalyzed rutaecarpine hydroxylation. Lines show the best fit as determined by nonlinear least-squares regression according to substrate inhibition as described under Materials and Methods. B, velocity () versus rutaecarpine concentration (S) plots of CYP1A1-catalyzed rutaecarpine hydroxylations at rutaecarpine concentration less than 10 µM. C, Lineweaver-Burk plots of CYP1A1-catalyzed rutaecarpine hydroxylations. Lines show the best fit as determined by nonlinear least-squares regression according to substrate inhibition. D, the plots of versus /S (Eadie-Hofstee plot). E, the plots of log(/Vmax – ) versus logS (Hill plot). The formation rates of 10-hydroxyrutaecarpine (), 11-hydroxyrutaecarpine (), 12-hydroxyrutaecarpine (), and 3-hydroxyrutaecarpine () were determined. Results represent the means of duplicate determinations.

View larger version (23K): [in this window] [in a new window]   FIG. 5. The velocity () versus rutaecarpine concentration (S) plots of CYP1A2 (A)- and CYP1B1 (B)-catalyzed rutaecarpine hydroxylation. The formation rates of 10-hydroxyrutaecarpine (), 11-hydroxyrutaecarpine (), 12-hydroxyrutaecarpine (), and 3-hydroxyrutaecarpine () were determined. Results represent the mean ± S.E.M. and means of three and two separate experiments with duplicate determinations, respectively.

View larger version (31K): [in this window] [in a new window]   FIG. 6. A and B, inhibitory kinetic analysis of the inhibition of CYP1B1-catalyzed 7-ethoxyresorufin O-deethylation activity by 10-hydroxyrutaecarpine. A, the velocity () versus rutaecarpine concentration (S) plots of CYP1A1-catalyzed rutaecarpine hydroxylation in the presence of 0 (), 0.1 (), and 0.2 () µM 10-hydroxyrutaecarpine. Solid lines represent the lines as determined by nonlinear regression analysis. B, the Lineweaver-Burke plot. Solid lines represent the lines as determined by linear regression analysis. C, binding spectra of 10-hydroxyrutaecarpine (1, 2, 3, 4, 5, and 7 µM) with CYP1B1 in 0.1 M potassium phosphate buffer, pH 7.4 at room temperature.

	Discussion

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Our previous report demonstrated that the alkaloid rutaecarpine was oxidized by rat liver microsomes to form 10-, 11-, 12-, and 3-hydroxyrutaecarpine (Ueng et al., 2005). In this report, our results revealed that rutaecarpine was also oxidized by human liver microsomal enzymes to form 10-, 11-, 12-, and 3-hydroxyrutaecarpine (Fig. 1). This is the first report to determine human metabolism of rutaecarpine in vitro. The results of microsomal inhibition were consistent with hydroxylation activities of bacterial membranes expressing bicistronic human P450s. Microsomal 10-hydroxylation activity was strongly inhibited by quinidine and ketoconazole (Table 1). Consistent with this inhibition result, E. coli-expressed human CYP2D6 and CYP3A4 had 10-hydroxylation activities higher than those of the other P450 enzymes (Table 2). Microsomal 12-hydroxylation was inhibited by -NF, quinidine, and ketoconazole. Human CYP1A2, CYP2C9, and CYP2D6 had detectable 12-hydroxylation activities (Table 2). Microsomal 11-hydroxylation was inhibited by -NF, quinidine, and ketoconazole. Human CYP1A2 and CYP2D6 had higher 11-hydroxylation activities than the other P450s. However, CYP3A4 had no detectable 11-hydroxylation activity. Microsomal 3-hydroxylation was inhibited by -NF, quinidine, and ketoconazole. 3-Hydroxylation activities of CYP2D6 and CYP3A4 were higher than those of the other P450s. However, CYP1A2 had relatively low 3-hydroxylation activity.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Rutaecarpine hydroxylations by human P450 forms.

Among P450s, our results showed that CYP1A1 had the highest rutaecarpine 10-, 11-, 12-, and 3-hydroxylation activities (Table 2). Our previous report demonstrated that rutaecarpine inhibited CYP1A1- and CYP1A2-catalyzed 7-ethoxyresorufin O-deethylation activities (Don et al., 2003). The IC₅₀ value of rutaecarpine for CYP1A2 was only 8% of that for CYP1A1 (Don et al., 2003). This strong CYP1A2 inhibition may result in relatively low rutaecarpine oxidation activity of CYP1A2 (Table 2). Kinetic analysis revealed that substrate inhibition occurred for both CYP1A1- and CYP1A2-catalyzed rutaecarpine hydroxylations (Figs. 4A and 5A). At a rutaecarpine concentration higher than 25 µM, hydroxylation activities decreased. In the in vitro systems, the pharmacologically active concentrations of rutaecarpine were in the range of 0.1 to 200 µM (Chen et al., 2002; Sheu, 1999). Substrate inhibition is possible at high pharmacologically active concentrations. The hydroxylations by CYP1A1 had K_i values 5- to 26-fold higher than K_m values. Substrate inhibition can be due to a variety of mechanisms including allosteric interaction and multiple active sites (Lin et al., 2001). For most P450-catalyzed oxidations, substrate inhibition was partial, suggesting that the substrate inhibition was due to the binding of a second molecule of substrate to the P450-substrate complex to form a poorly productive or nonproductive enzyme complex and reduce the oxidation rates (Lin et al., 2001). Consistent with this allosteric interaction in substrate inhibition, rutaecarpine strongly inhibited CYP1A2-catalyzed 7-ethoxyresorufin O-deethylation activity in a mixed type of noncompetitive and uncompetitive inhibition. The K_i value for CYP1A2-substrate complex was less than that for CYP1A2 only, suggesting a strong binding of rutaecarpine to CYP1A2-substrate complex (Y. F. Ueng, S. Y. Wang, and M. J. Don, unpublished result). Besides CYP1A1 and CYP1A2, rutaecarpine also inhibited CYP1B1-catalyzed 7-ethoxyresorufin O-deethylation noncompetitively with an IC₅₀ value about 3-fold higher than that for CYP1A2, suggesting that rutaecarpine bound to CYP1B1 and CYP1B1-substrate complexes with similar dissociation constants (Don et al., 2003). However, CYP1B1 did not show the substrate inhibition as obviously as CYP1A1 and CYP1A2.

10-Hydroxyrutaecarpine was the most abundant metabolite formed by most human liver samples. Among CYP1 members, CYP1A1 and CYP1B1 oxidized rutaecarpine to form mainly 10-hydroxyrutaecarpine. For CYP1A1, the plot of velocity versus substrate concentration at the concentration less than 10 µM showed a sigmoidal pattern (Fig. 4B). Eadie-Hofstee and Hill plots showed a kinetic pattern with cooperativity higher than 1 for CYP1A1-catalyzed rutaecarpine 10-, 11-, 12-, and 3-hydroxylations (Fig. 4, D and E). The positive cooperativity seen in the sigmoidal velocity versus substrate concentration plot could be attributed to several factors. These factors include the presence of more than one substrate binding site or conformation in CYP1A1 and/or product inhibition (Duggleby, 1994). Thus, the effect of 10-hydroxyrutaecarpine on CYP1 members was studied. 10-Hydroxyrutaecarpine strongly inhibited CYP1A1-, CYP1A2-, and CYP1B1-catalyzed 7-ethoxyresorufin O-deethylation activities with the IC₅₀ values in the order of CYP1A1 CYP1A2 > CYP1B1. The inhibition of CYP1 by this oxidation product may shift the velocity versus substrate concentration plot to a sigmoidal pattern. In the future, it will be of interest to determine the plasma concentrations of 10-hydroxyrutaecarpine and/or its conjugates to assess the role of 10-hydroxyrutaecarpine in drug interactions of the rutaecarpine-containing remedy in our previous report (Ueng et al., 2003).

The major finding is that multiple P450 enzymes, including CYP1A2, CYP2D6, and CYP3A4 participated in human liver microsomal rutaecarpine hydroxylation (Fig. 7). Genetic variations of CYP1A2, CYP2D6, and CYP3A4 have been reported in humans (Hasler et al., 1999). To clarify the possible individual differences in pharmacological response to E. rutaecarpa due to differential metabolic capacity, it will be of interest to investigate the pharmacological effects of rutaecarpine metabolites. The main pharmacological targets of rutaecarpine are the cardiovascular and circulation systems. The pharmacological effects of rutaecarpine may show tissue specificity. Among the P450 enzymes studied here, CYP1A1 had 10-, 11-, 12-, and 3-hydroxylation activities 6 to 26-, 6 to 9-, 10 to 16-, and 7 to 27-fold higher than the activities of the other P450s, respectively. CYP1A1 is essentially extrahepatic and is inducible in the endothelial cells of blood vessels (Dey et al., 1999; Ding and Kaminsky, 2003). Therefore, CYP1A1 may also play an important role in the toxicity and pharmacological activities of rutaecarpine in extrahepatic tissues.

	Acknowledgments

 
We thank Dr. Ta-Liang Chen and Dr. F. Peter Guengerich for providing human liver samples.

	Footnotes

 
This work was supported by a National Science Council grant NSC 91-2320-B077-010 and National Research Institute of Chinese Medicine, Taiwan.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.007849.

ABBREVIATIONS: P450, cytochrome P450; -NF, -naphthoflavone; G6P, glucose 6-phosphate; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. Yune-Fang Ueng, National Research Institute of Chinese Medicine, 155–1, Li-Nong Street, Sec. 2, Taipei 112, Taiwan, R.O.C. E-mail: ueng{at}nricm.edu.tw

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