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Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver
Comparative Biochemistry and Physiology Part C 129 Ž2001. 361᎐368
Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver Xiuzohng Wua , Atsuko Nodaa , Hiroshi Nodab, Yorishige Imamurac,U b
a Faculty of Pharmaceutical Sciences, Kyushu Uni¨ ersity, 3-3-1, Maidashi, Higashi-ku, Fukuoka 812-0054, Japan Department of Hospital Pharmacy, School of Medicine, Uni¨ ersity of Occupational and En¨ ironmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-0804, Japan c Faculty of Pharmaceutical Sciences, Kumamoto Uni¨ ersity, 5-1, Oe-honmachi, Kumamoto 862-0973, Japan
Received 2 March 2001; received in revised form 18 May 2001; accepted 24 May 2001
Abstract The further metabolism of N-desisopropylpropranolol ŽNDP., a side-chain metabolite of propranolol ŽPL., was investigated in isolated rat hepatocytes. Propranolol glycol ŽPGL. was generated from NDP as a major metabolite. Naphtetrazole ŽNTE., a potent inhibitor of monoamine oxidase ŽMAO., significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde Ž4-NBA., a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP w SŽy.-form of NDPx was larger than that of PGL formed from R-NDP w RŽq.-form of NDPx. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Propranolol metabolism; Monoamine oxidase; Aldehyde reductase; Side-chain metabolism; Aldehyde intermediate; Rat hepatocyte; Rat liver mitochondria; Rat liver cytosol; Enantioselective metabolism
U
Corresponding author. E-mail address: [email protected] ŽY. Imamura..
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 1 . 0 0 2 1 2 - 5
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1. Introduction Propranolol ŽPL. is an adrenergic -blocker and widely used in the treatment of cardiovascular disorders and hypertension. Major metabolic pathways of PL consist of naphthalene ringhydroxylation, side-chain N-desisopropylation and side-chain glucuronidation in animals and humans ŽWalle and Gaffney, 1972; Bargar et al., 1983; Walle et al., 1985, 1988.. Interestingly, 4-hydroxypropranolol is known to be a pharmacologically active metabolite ŽRoutlege and Shand, 1979.. N-Desisopropylpropranolol ŽNDP., which is generated from PL by the side-chain N-desisopropylation, is subsequently metabolized to propranolol glycol ŽPGL., naphthoxylactic acid ŽNLA. and naphthoxyacetic acid ŽNAA. through an aldehyde intermediate ŽWalle and Gaffney, 1972; Walle et al., 1985., and metabolized to acetyl conjugate of NDP ŽAcNDP. ŽNoda et al., 1994., as evident from the metabolic pathways in Fig. 1. Recently, the oxidative metabolism of PL to NDP has been reported to be catalyzed mainly by one of cytochrome P450 ŽCYP. isozymes,
CYP1A2, in human liver ŽMasubuchi et al., 1994; Yoshimoto et al., 1995.. However, information on the enzymes responsible for the metabolism of NDP to PGL or NLA has been very limited, probably due to the chemical instability of the aldehyde intermediate generated during the further metabolism of NDP. It is possible to assume that NDP is deaminated to the aldehyde intermediate by monoamine oxidase ŽMAO. ŽEC 1.4.3.4., and that the formed aldehyde intermediate is rapidly reduced to PGL by enzymes such as aldehyde reductase ŽEC 1.1.1.2., aldose reductase ŽEC 1.1.1.21. and carbonyl reductase ŽEC 1.1.1.184., or oxidized to NLA by aldehyde dehydrogenase ŽEC 1.2.1.3. ŽWermuth and Munch, 1979; Opper¨ mann and Maser, 2000; Vasiliou et al., 2000.. In the present study, we attempt to synthesize the side-chain metabolites of propranolol, NDP and PGL, according to the method of Walle and Gaffney Ž1972., and provide evidence that both MAO and aldehyde reductase are involved in the metabolism of NDP to PGL in rat liver.
2. Materials and methods 2.1. Materials NLA and NAA, metabolites of PL, were purchased from Cambridge Research Biochemicals ŽCheshire, UK. and Wako Pure Chemical Industries ŽOsaka, Japan., respectively. Phenobarbital Žsodium salt. was obtained from Tokyo Kasei Kogyo Co. ŽTokyo, Japan., and 4-nitrobenzaldehyde Ž4-NBA. was obtained from the Sigma Chemical Co. ŽSt. Louis, MO, USA.. NADPH and NADH were the products of the Oriental Yeast Co. ŽTokyo, Japan .. Tetrazolo w5,1axphthalazine ŽTetra-P. and 3-ethyl-s-triazolow3,4a xphthalazine ŽETP., and 1,2,3-Triazolow1,5axquinoline ŽTri-Q. were synthesized by the method of Haegele et al. Ž1976., and by the method of Abramovitch and Takaya Ž1972. with some modifications, respectively. Naphtetrazole ŽNTE. was synthesized as described previously ŽKai et al., 1988.. All other chemicals were of reagent grade. 2.2. Syntheses of metabolites
Fig. 1. Metabolic pathway of PL to NDP, PGL, NLA, NAA and AcNDP.
Ž1. NDP. This metabolite Žracemate. was synthesized from 1-Ž1-naphthoxy.-2,3-epoxypropane
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via the phthalimide intermediate according to the method of Walle and Gaffney Ž1972.; m.p. 208᎐209⬚C. MS: mrz 217 ŽMq. . Anal. calcd. for C 13 H 15 NO 2 ⭈ HCl: C, 61.50; H, 6.31; N, 5.52. Found: C, 61.60; H, 6.31; N, 5.29. 1-Ž1Naphthoxy.-2,3-epoxypropnane was synthesized from 1-naphthol and 1-bromo-2,3-epoxypropane with careful protection from oxygen and light. RŽq.- and SŽy.-enantiomers Ž R-NDP and SNDP. of NDP were separated from the racemate by HPLC using a Chiracel OD column ŽTosoh, Tokyo, Japan.. The specific optical rotations in methanol of R-NDP and S-NDP were q3.59⬚ and y3.59⬚, respectively. Ž2. PGL. This metabolite was synthesized by hydrolyzing 1-Ž1-naphthoxy.2,3-epoxy-propane in a 1:1 mixture of 1 N HCl and tetrahydrofuran according to the method of Walle and Gaffney Ž1972.; m.p. 68᎐70⬚C. FABrMS: mrz 218 ŽMq. . Anal. calcd. for C 13 H 14 O 3 : C, 71.60; H, 6.42. Found: C, 71.35; H, 6.50. Ž3. AcNDP. The synthesis of this metabolite was as described previously ŽNoda et al., 1994.. NDP was added to the mixed solution of acetic anhydride and pyridine, and the obtained diacetylate was hydrolyzed with 1 N NaOH at 70⬚C for 15 min to give the N-monoacetylate ŽAcNDP.. Crude AcNDP was recrystallized from methanol including a small amount of isopropyl ether; m.p. 101᎐102⬚C. MS: mrz 259 ŽMq. . Anal. calcd. for C 15 H 17 NO 3 : C, 69.45; H, 6.60; N, 5.47. Found: C, 69.48; H, 6.61; N, 5.40.
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taining substrate wNDP Žracemate., R-NDP or S-NDPx at various concentrations, 12.6 mM N-2hydroxyethylpiperazine-N-ethane sulfonic acid ŽHEPES. and 10 mM glucose in rotating roundbottomed flasks at 37⬚C, during continuous gassing with 95%O 2 ᎐5%CO 2 . After incubation for 10, 20 and 30 min, aliquots Žeach 1.0 ml. of the medium were taken, and NDP and its metabolites ŽPGL, AcNDP, NLA and NAA. were determined by HPLC as described below. 2.5. Preparation of subcellular fractions and incubation
Male Wistar rats Ž200᎐260 g. were purchased from Kuroda Pure Animals ŽKumamoto, Japan.. Animals were raised under controlled lighting Ž06:00᎐18:00 h., temperature Ž20 " 2⬚C. and humidity Ž50 " 10%., and had free access to a standard laboratory diet and water. All animal experiments were undertaken in obedience to the guideline principles for the care and use of laboratory animals.
Animals were killed by decapitation. Hepatic subcellular fractions Žmitochondria, microsomes and cytosol. were prepared by standard procedures. After perfusion with ice-cold 250 mM sucrose solution, the livers were immediately removed and homogenized with 10 mM Tris᎐HCl buffer ŽpH 7.5. containing 250 mM sucrose and 10 mM EDTA, using a Potter᎐Elvehjem homogenizer. All subsequent procedures were performed at 3᎐5⬚C. The homogenate was centrifuged at 600 = g for 10 min to sediment the nuclei and cell debris. The resulting supernatant was centrifuged at 9000 = g for 20 min to sediment mitochondria. The mitochondrial pellets were resuspended and then centrifuged at 15 000 = g for 5 min. Furthermore, the 9000 = g supernatant was centrifuged at 105 000 = g for 60 min to obtain the microsomal pellets and cytosolic fraction. The microsomal pellets were resuspended and then centrifuged at 105 000 = g for 60 min. The reaction mixture consisted of 100 mM potassium phosphate buffer ŽpH 7.4., 500 M cofactor ŽNADPH or NADH., substrate ŽNDP. at various concentrations, and enzyme preparation Žmitochondria, cytosol, microsomes, or mixture of mitochondria and cytosol., in a total volume of 2.0 ml. After incubation at 37⬚C for 15 min, aliquots Ž1.0 ml. of the reaction mixtures were taken and PGL formed was determined by HPLC as described below.
2.4. Preparation of hepatocytes and incubation
2.6. HPLC analysis
Animals were killed by decapitation. Hepatocytes were carefully prepared from rat livers according to the method of Moldeus ´ et al. Ž1978.. The cell concentration of the hepatocytes was adjusted to 8 = 10 6 cellsrml. Incubation was carried out in Krebs᎐Henseleit buffer ŽpH 7.4. con-
To 1.0 ml of the incubated samples, 0.1 N Na 2 CO 3 Ž0.3 ml. and methanol Ž0.1 ml. containing Tetra-P and Tri-Q as the internal standard were added, and the mixture was extracted with 3.0 ml of chloroform. The organic layer was removed and evaporated to dryness. The residue
2.3. Animals
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was dissolved in methanol Ž0.1 ml. and subjected to HPLC for the determination of NDP, AcNDP and PGL. The aqueous layer was acidified by adding 2 N HCl Ž1.0 ml. containing ETP as the internal standard and extracted with 4.0 ml of ethyl acetate. The organic layer was removed and evaporated to dryness. The residue was dissolved in methanol Ž0.1 ml. and subjected to HPLC for the determination of NLA and NAA. HPLC was carried out using a Shimadzu LC-10A apparatus equipped with a reversed phase column ŽTSK gel ODS-80 TM, 25 cm = 4.6 mm, Tosoh, Tokyo, Japan . and a spectrofluorometric detector ŽShimadzu RF-550.. The column eluate was monitored at 310 nm Žexcitation. and 380 nm Žemission.. The column temperature was 37⬚C and the flow rate was 0.8 mlrmin. The mobile phase was a mixture of acetonitrilermethanolrwaterracetic acid Ž18:7:75:1.. 2.7. Determination of protein content Protein content was determined by using the BIO-RAD protein assay kit ŽBIO-RAD, Hercules, CA.. 2.8. Statistical analysis Data were expressed as the means " S.D. of three experiments. The statistical significance of differences was analyzed by Student’s t-test, and P- 0.05 was considered significant.
3. Results 3.1. Metabolism of NDP in isolated rat hepatocytes The further metabolism of NDP, a side-chain metabolite of PL, was examined in isolated rat hepatocytes. As shown in Fig. 2a, a rapid disappearance of NDP from the incubation medium was observed. Furthermore, two major metabolites, NLA and PGL, were generated from NDP, accompanied by small amounts of NAA and AcNDP ŽFig. 2b.. However, there was a large difference between the amounts of NDP remaining and these four metabolites formed Žsee Fig. 2a,b., indicating the formation of metabolites other than NLA, PGL, NAA and AcNDP. We also examined the effect of NTE, which is a potent inhibitor of MAO found by us ŽKai et al.,
Fig. 2. Disappearance of NDP from the incubation medium of isolated rat hepatocyte Ža. and formation of various metabolites from NDP in isolated rat hepatocyte Žb.. NDP Ž500 M. was used as the substrate. Each point represents the mean " S.D. of three experiments. Žb. ', NLA; I, PGL; 䢇, NAA; ^, AcNDP.
1985., on the disappearance of NDP from the incubation medium. The inhibitory potency against MAO of NTE is known to be comparable to those of typical MAO inhibitors, iproniazid and nialamide ŽKai et al., 1985, 1988.. As expected, NTE significantly retarded the disappearance of NDP from the incubation medium ŽFig. 3.. This finding suggests that NTE decreases the formation of the aldehyde intermediate from NDP by inhibiting MAO. 3.2. Metabolism of NDP in mixture of rat li¨ er mitochondria and cytosol with NADPH Fig. 4 shows the effects of phenobarbital and
X. Wu et al. r Comparati¨ e Biochemistry and Physiology Part C 129 (2001) 361᎐368
Fig. 3. Effect of NTE on the disappearance of NDP from the incubation medium of isolated rat hepatocytes. The concentrations of NDP and NTE used as the substrate and inhibitor, respectively, were 100 M. Each point represents the mean " S.D. of three experiments. I, control; `, in the presence of NTE. U P- 0.05, UUU P- 0.001, significantly different from control value in the corresponding time.
4-NBA on the formation of PGL from NDP in a mixture of rat liver mitochondria and cytosol with NADPH Žmitochondria q cytosol with NADPH.. Phenobarbital and 4-NBA were used as a specific inhibitor of aldehyde reductase ŽTurner and Hick, 1976; Felsted et al., 1977; Ansari et al., 1991. and as a substrate inhibitor because of a typical substrate of aldehyde reductase ŽDaly and Mantle, 1982; Ellis and Hayes, 1995., respectively. These two compounds significantly decreased the PGL formation. Furthermore, the effect of 4-NBA on the PGL formation was kinetically analyzed. As evident from the Dixon-plots, 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation ŽFig. 5.; 4-NBA was confirmed to be reduced to 4-nitrobenzyl alcohol in rat liver cytosol with NADPH Ždata not shown.. The pH dependency for the formation of PGL from NDP was also examined in the reaction mixture. As shown in Fig. 6, the pH optimum of the PGL formation was observed at approximately 8.0.
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Fig. 4. Effects of phenobarbital ŽPB. and 4-NBA on the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH. NDP Ž100 M. was used as the substrate. Each bar, expressed as the percentage of control, represents the mean " S.D. of three experiments. UU P - 0.01, UUU P - 0.001, significantly different from control value.
indicating the enantioselective metabolism of NDP to PGL.
4. Discussion The present study demonstrated that PGL is generated from NDP as a major metabolite in isolated rat hepatocytes. The metabolic reaction of NDP to PGL probably proceeds through an aldehyde intermediate generated from NDP by MAO. This was supported from the fact that the
3.3. Enantioselecti¨ e metabolism of NDP in isolated rat hepatocytes The metabolism of R-NDP and S-NDP to PGL was compared in isolated rat hepatocytes. The amount of PGL formed from S-NDP was larger than that of PGL formed from R-NDP ŽFig. 7.,
Fig. 5. Dixon-plots for the inhibition by 4-NBA of PGL formation in the mixture of rat liver mitochondria and cytosol with NADPH. Velocity Ž V . is expressed as nmolrminrmg protein. The concentrations of NDP used as the substrate were 10 M ŽB., 20 M Ž䢇. and 40 M Ž'..
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Fig. 6. pH dependency for the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH. NDP Ž100 M. was used as the substrate. Each point represents the mean " S.D. of three experiments.
PGL formation is decreased by adding NTE, a potent inhibitor of MAO, to the incubation medium. To establish the characteristics of enzymes responsible for the metabolism of NDP to PGL in rat liver, a preliminary study was carried out by using subcellular fractions Žmitochondria, cytosol, microsomes and their mixtures. prepared from rat liver Ždata not shown.. PGL was most efficiently formed from NDP in a mixture of mitochondria and cytosol with NADPH. On the other hand, PGL was little formed from NDP when NADPH was replaced by NADH, and in
Fig. 7. Formation of PGL from R-NDP or S-NDP in isolated rat hepatocytes. R-NDP Ž100 M. or S-NDP Ž100 M. was used as the substrate. Each point represents the mean " S.D. of three experiments. `, from R-NDP; I, from S-NDP. UU P- 0.01, UUU P- 0.001, significantly different from the amount of PGL formed from R-NDP in the corresponding time.
the enzyme reaction system of mitochondria alone, microsomes alone or cytosol with NADPH. These results lead us to suggest that in addition to MAO Žmitochondrial enzyme., an NADPHdependent cytosolic enzyme is involved in the metabolism of NDP to PGL in rat liver. In this study, we revealed that phenobarbital and 4-NBA significantly decrease the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH, and that 4-NBA is a competitive inhibitor of the enzyme responsible for the PGL formation, based on the kinetic data of Dixon-plots. Phenobarbital is a specific inhibitor of aldehyde reductase ŽTurner and Hick, 1976; Felsted et al., 1977; Ansari et al., 1991. and 4-NBA is widely used as a substrate of aldehyde reductase ŽDaly and Mantle, 1982; Ellis and Hayes, 1995.. Thus, it is reasonable to assume that the metabolic reduction of the aldehyde intermediate to PGL is catalyzed by aldehyde reductase. More recently, it has been reported that aldose reductase, which is a cytosolic enzyme like aldehyde reductase, mainly contributes to the metabolism of norepinephrine to 3,4-dihydroxyphenylglycol in the presence of MAO ŽKawamura et al., 1999.. Aldose reductase and carbonyl reductase also have the ability to reduce aldehydes. However, aldose reductase and carbonyl reductase are not inhibited by phenobarbital ŽAhmed et al., 1981; Ansari et al., 1991., suggesting that aldehyde reductase rather than these two enzymes is mainly involved in the metabolic reduction of the aldehyde intermediate to PGL. Aldehyde reductase purified from rat liver has an optimal pH of 8.5 when daunorubicin, an effective anticancer drug, is used as a substrate ŽFelsted et al., 1977.. A similar pH optimum is observed for daunorubicin reduction catalyzed by rabbit liver aldehyde reductase ŽAhmed et al., 1981.. On the other hand, aldose reductase and carbonyl reductase have their maximum activity in the range of pH 6.0᎐6.5 ŽPropper and Maser, ¨ 1997.. In this study, the optimal pH for the formation of PGL from NDP was approximately 8.0 in the reaction mixture of rat liver mitochondria and cytosol with NADPH. This result also supports the idea that the metabolism of the aldehyde intermediate to PGL is mainly mediated by aldehyde reductase. So far, little attempt has been made to evaluate the contribution of aldehyde reductase to drug metabolism. This is because,
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among clinically used drugs, there is no drug having an aldehyde group within its chemical structure. However, aldehyde intermediates are known to be generated as metabolites of endogenous and exogeneous compounds including drugs. It is possible that aldehyde intermediates are highly reactive molecules and induce a variety of toxic effects. We propose the importance of aldehyde reductase as one of drug-metabolizing enzymes. Fujita et al. Ž1993. have shown the enantioselectivity for the oxidation Ž4-, 5- and 7-hydroxylations and N-desisopropylation. of propranolol by CYPs purified from rat liver. Species difference in the enantioselective oxidation of propranolol is also examined ŽNarimatsu et al., 2000.. However, enantioselectivity for the further metabolism of NDP generated by the N-desisopropylation of propranolol remains to be elucidated. We found that in isolated rat hepatocytes, the amount of PGL formed from S-NDP is larger than that of PGL formed from R-NDP. Either MAO or aldehyde reductase may play a critical role in the enantioselective metabolism of NDP to PGL. Further studies are in progress to establish the enzymology for the enantioselective metabolism of NDP to PGL in rat liver. In conclusion, the data obtained in the present study demonstrate that in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. References Abramovitch, R.A., Takaya, T., 1972. The reaction of sulfonyl azides with pyridines and fused pyridine derivatives. J. Org. Chem. 37, 2022᎐2029. Ahmed, N.K., Felsted, R.L., Bachur, N.R., 1981. Daunorubicin reduction mediated by aldehyde and ketone reductases. Xenobiotica 11, 131᎐136. Ansari, N.H., Bhatnagar, A., Liu, S., Srivastava, S.K., 1991. Purification and characterisation of aldose reductase and aldehyde reductase from human kidney. Biochem. Int. 25, 755᎐765. Bargar, E.M., Walle, U.K., Bai, S.A., Walle, T., 1983. Quantitative metabolic fate of propranolol in the dog, rat and hamster using radiotracer, high performance liquid chromatography, and gas chromatography᎐mass spectrometry techniques. Drug Metab. Dispos. 11, 266᎐272.
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Daly, A.K., Mantle, T.J., 1982. Purification and characterisation of the multiple forms of aldehyde reductase in ox kidney. Biochem. J. 205, 373᎐380. Ellis, E.M., Hayes, J.D., 1995. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase. Biochem. J. 312, 535᎐541. Felsted, R.L., Richter, D.R., Bachur, N.R., 1977. Rat liver aldehyde reductase. Biochem. Pharmacol. 26, 1117᎐1124. Fujita, S., Umeda, S., Funae, Y. et al., 1993. Regio- and stereoselective propranolol metabolism by 15 forms of purified cytochrome P-450 from rat liver. J. Pharmacol. Exp. Ther. 264, 226᎐233. Haegele, K.D., Skrdlant, H.B., Robie, N.W., Lalka, D., MaNay Jr., J.L., 1976. Determination of hydralazine and its metabolites by gas chromatography-mass spectrometry. J. Chromatogr. 126, 517᎐534. Kai, M., Noda, A., Noda, H., Goto, S., 1985. Detection of a new series of monoamine oxidase inhibitors. Chem. Pharm. Bull. 33, 5585᎐5588. Kai, M., Noda, A., Noda, H., Goto, S., 1988. Structure᎐activity relationship of a new series of tricyclic monoamine oxidase inhibitors of pentanthrene type. Chem. Pharm. Bull. 36, 3604᎐3608. Kawamura, M., Eisenhofer, G., Kopin, I.J. et al., 1999. Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats. Biochem. Pharmacol. 58, 517᎐524. Masubuchi, Y., Hosokawa, S., Horie, T. et al., 1994. Cytochrome P450 isozymes involved in propranolol metabolism in human liver microsomes. The role of CYP2D6 as ring-hydroxylase and CYP1A2 as N-desisopropylase. Drug Metab. Dispos. 22, 909᎐915. Moldeus, J., Orrenius, S., 1978. Isolation ´ P., Hogberg, ¨ and use of liver cells. In: Fleisher, S., Packer, L. ŽEds.., Methods in Enzymology, 52. Academic Press, New York, pp. 60᎐71. Narimatsu, S., Kobayashi, N., Masubuchi, Y. et al., 2000. Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes. Chem. Biol. Interact. 127, 73᎐90. Noda, A., Ono, Y., Inokuchi, K. et al., 1994. Detection of 1-acetamino-3-Ž1-naphthyloxy.-2-propanol as a new metabolite of propranolol. Biol. Pharm. Bull. 17, 1433᎐1435. Oppermann, U.C., Maser, E., 2000. Molecular and structural aspects of xenobiotic carbonyl metabolizing enzymes. Role of reductases and dehydrogenases in xenobiotic phase I reactions. Toxicology 144, 71᎐81. Propper, D., Maser, E., 1997. Carbonyl reduction of ¨ daunorubicin in rabbit liver and heart. Pharmacol. Toxicol. 80, 240᎐245. Routlege, P.A., Shand, D.G., 1979. Clinical pharmacokineics of propranolol. Clin. Pharmacokinet. 4, 73᎐90.
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X. Wu et al. r Comparati¨ e Biochemistry and Physiology Part C 129 (2001) 361᎐368
Turner, A.J., Hick, P.E., 1976. Metabolism of daunorubicin by a barbiturate-sensitive aldehyde reductase from rat liver. Biochem. J. 159, 819᎐822. Vasiliou, V., Pappa, A., Petersen, D.R., 2000. Role of aldehyde dehydrogenase in endogenous and xenobiotic metabolism. Chem. Biol. Interact. 129, 1᎐19. Walle, T., Gaffney, T.E., 1972. Propranolol metabolism in man and dog: mass spectrometric identification of six new metabolites. J. Pharmacol. Exp. Ther. 182, 83᎐92. Walle, T., Walle, U.K., Olanoff, L.S., 1985. Quantitative account of propranolol metabolism in urine of normal man. Drug Metab. Dispos. 13, 204᎐209. Walle, T., Webb, J.G., Bagwell, E.E., Walle, U.K.,
Daniell, H.B., Gaffney, T.E., 1988. Stereoselective delivery and action of beta receptor antagonists. Biochem. Pharmacol. 37, 115᎐124. Wermuth, B., Munch, J.D.B., 1979. Reduction of bio¨ genic aldehydes by aldehyde reductase and alcohol dehydrogenase from human liver. Biochem. Pharmacol. 28, 1431᎐1433. Yoshimoto, K., Echizen, H., Chiba, K., Tani, M., Ishizaki, T., 1995. Identification of human CYP isoforms involved in the metabolism of propranolol enantiomers. N-Desisopropylation is mediated mainly by CYP1A2. Brit. J. Clin. Pharmacol. 39, 421᎐431.
Side-chain metabolism of propranolol: involvement of monoamine oxidase and aldehyde reductase in the metabolism of N-desisopropylpropranolol to propranolol glycol in rat liver Xiuzohng Wua , Atsuko Nodaa , Hiroshi Nodab, Yorishige Imamurac,U b
a Faculty of Pharmaceutical Sciences, Kyushu Uni¨ ersity, 3-3-1, Maidashi, Higashi-ku, Fukuoka 812-0054, Japan Department of Hospital Pharmacy, School of Medicine, Uni¨ ersity of Occupational and En¨ ironmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu 807-0804, Japan c Faculty of Pharmaceutical Sciences, Kumamoto Uni¨ ersity, 5-1, Oe-honmachi, Kumamoto 862-0973, Japan
Received 2 March 2001; received in revised form 18 May 2001; accepted 24 May 2001
Abstract The further metabolism of N-desisopropylpropranolol ŽNDP., a side-chain metabolite of propranolol ŽPL., was investigated in isolated rat hepatocytes. Propranolol glycol ŽPGL. was generated from NDP as a major metabolite. Naphtetrazole ŽNTE., a potent inhibitor of monoamine oxidase ŽMAO., significantly retarded the disappearance of NDP from the incubation medium, suggesting the involvement of MAO in the deamination of NDP to an aldehyde intermediate. In a reaction mixture of rat liver mitochondria and cytosol with NADPH, phenobarbital, a specific inhibitor of aldehyde reductase, and 4-nitrobenzaldehyde Ž4-NBA., a substrate inhibitor of aldehyde reductase, decreased the formation of PGL from NDP. 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation. The optimal pH for the formation of PGL from NDP in the reaction mixture was approximately 8.0. Based on these results, we propose the possibility that, in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. Furthermore, the enantioselective metabolism of NDP to PGL was examined. In isolated rat hepatocytes, the amount of PGL formed from S-NDP w SŽy.-form of NDPx was larger than that of PGL formed from R-NDP w RŽq.-form of NDPx. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Propranolol metabolism; Monoamine oxidase; Aldehyde reductase; Side-chain metabolism; Aldehyde intermediate; Rat hepatocyte; Rat liver mitochondria; Rat liver cytosol; Enantioselective metabolism
U
Corresponding author. E-mail address: [email protected] ŽY. Imamura..
1532-0456r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 1 . 0 0 2 1 2 - 5
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1. Introduction Propranolol ŽPL. is an adrenergic -blocker and widely used in the treatment of cardiovascular disorders and hypertension. Major metabolic pathways of PL consist of naphthalene ringhydroxylation, side-chain N-desisopropylation and side-chain glucuronidation in animals and humans ŽWalle and Gaffney, 1972; Bargar et al., 1983; Walle et al., 1985, 1988.. Interestingly, 4-hydroxypropranolol is known to be a pharmacologically active metabolite ŽRoutlege and Shand, 1979.. N-Desisopropylpropranolol ŽNDP., which is generated from PL by the side-chain N-desisopropylation, is subsequently metabolized to propranolol glycol ŽPGL., naphthoxylactic acid ŽNLA. and naphthoxyacetic acid ŽNAA. through an aldehyde intermediate ŽWalle and Gaffney, 1972; Walle et al., 1985., and metabolized to acetyl conjugate of NDP ŽAcNDP. ŽNoda et al., 1994., as evident from the metabolic pathways in Fig. 1. Recently, the oxidative metabolism of PL to NDP has been reported to be catalyzed mainly by one of cytochrome P450 ŽCYP. isozymes,
CYP1A2, in human liver ŽMasubuchi et al., 1994; Yoshimoto et al., 1995.. However, information on the enzymes responsible for the metabolism of NDP to PGL or NLA has been very limited, probably due to the chemical instability of the aldehyde intermediate generated during the further metabolism of NDP. It is possible to assume that NDP is deaminated to the aldehyde intermediate by monoamine oxidase ŽMAO. ŽEC 1.4.3.4., and that the formed aldehyde intermediate is rapidly reduced to PGL by enzymes such as aldehyde reductase ŽEC 1.1.1.2., aldose reductase ŽEC 1.1.1.21. and carbonyl reductase ŽEC 1.1.1.184., or oxidized to NLA by aldehyde dehydrogenase ŽEC 1.2.1.3. ŽWermuth and Munch, 1979; Opper¨ mann and Maser, 2000; Vasiliou et al., 2000.. In the present study, we attempt to synthesize the side-chain metabolites of propranolol, NDP and PGL, according to the method of Walle and Gaffney Ž1972., and provide evidence that both MAO and aldehyde reductase are involved in the metabolism of NDP to PGL in rat liver.
2. Materials and methods 2.1. Materials NLA and NAA, metabolites of PL, were purchased from Cambridge Research Biochemicals ŽCheshire, UK. and Wako Pure Chemical Industries ŽOsaka, Japan., respectively. Phenobarbital Žsodium salt. was obtained from Tokyo Kasei Kogyo Co. ŽTokyo, Japan., and 4-nitrobenzaldehyde Ž4-NBA. was obtained from the Sigma Chemical Co. ŽSt. Louis, MO, USA.. NADPH and NADH were the products of the Oriental Yeast Co. ŽTokyo, Japan .. Tetrazolo w5,1axphthalazine ŽTetra-P. and 3-ethyl-s-triazolow3,4a xphthalazine ŽETP., and 1,2,3-Triazolow1,5axquinoline ŽTri-Q. were synthesized by the method of Haegele et al. Ž1976., and by the method of Abramovitch and Takaya Ž1972. with some modifications, respectively. Naphtetrazole ŽNTE. was synthesized as described previously ŽKai et al., 1988.. All other chemicals were of reagent grade. 2.2. Syntheses of metabolites
Fig. 1. Metabolic pathway of PL to NDP, PGL, NLA, NAA and AcNDP.
Ž1. NDP. This metabolite Žracemate. was synthesized from 1-Ž1-naphthoxy.-2,3-epoxypropane
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via the phthalimide intermediate according to the method of Walle and Gaffney Ž1972.; m.p. 208᎐209⬚C. MS: mrz 217 ŽMq. . Anal. calcd. for C 13 H 15 NO 2 ⭈ HCl: C, 61.50; H, 6.31; N, 5.52. Found: C, 61.60; H, 6.31; N, 5.29. 1-Ž1Naphthoxy.-2,3-epoxypropnane was synthesized from 1-naphthol and 1-bromo-2,3-epoxypropane with careful protection from oxygen and light. RŽq.- and SŽy.-enantiomers Ž R-NDP and SNDP. of NDP were separated from the racemate by HPLC using a Chiracel OD column ŽTosoh, Tokyo, Japan.. The specific optical rotations in methanol of R-NDP and S-NDP were q3.59⬚ and y3.59⬚, respectively. Ž2. PGL. This metabolite was synthesized by hydrolyzing 1-Ž1-naphthoxy.2,3-epoxy-propane in a 1:1 mixture of 1 N HCl and tetrahydrofuran according to the method of Walle and Gaffney Ž1972.; m.p. 68᎐70⬚C. FABrMS: mrz 218 ŽMq. . Anal. calcd. for C 13 H 14 O 3 : C, 71.60; H, 6.42. Found: C, 71.35; H, 6.50. Ž3. AcNDP. The synthesis of this metabolite was as described previously ŽNoda et al., 1994.. NDP was added to the mixed solution of acetic anhydride and pyridine, and the obtained diacetylate was hydrolyzed with 1 N NaOH at 70⬚C for 15 min to give the N-monoacetylate ŽAcNDP.. Crude AcNDP was recrystallized from methanol including a small amount of isopropyl ether; m.p. 101᎐102⬚C. MS: mrz 259 ŽMq. . Anal. calcd. for C 15 H 17 NO 3 : C, 69.45; H, 6.60; N, 5.47. Found: C, 69.48; H, 6.61; N, 5.40.
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taining substrate wNDP Žracemate., R-NDP or S-NDPx at various concentrations, 12.6 mM N-2hydroxyethylpiperazine-N-ethane sulfonic acid ŽHEPES. and 10 mM glucose in rotating roundbottomed flasks at 37⬚C, during continuous gassing with 95%O 2 ᎐5%CO 2 . After incubation for 10, 20 and 30 min, aliquots Žeach 1.0 ml. of the medium were taken, and NDP and its metabolites ŽPGL, AcNDP, NLA and NAA. were determined by HPLC as described below. 2.5. Preparation of subcellular fractions and incubation
Male Wistar rats Ž200᎐260 g. were purchased from Kuroda Pure Animals ŽKumamoto, Japan.. Animals were raised under controlled lighting Ž06:00᎐18:00 h., temperature Ž20 " 2⬚C. and humidity Ž50 " 10%., and had free access to a standard laboratory diet and water. All animal experiments were undertaken in obedience to the guideline principles for the care and use of laboratory animals.
Animals were killed by decapitation. Hepatic subcellular fractions Žmitochondria, microsomes and cytosol. were prepared by standard procedures. After perfusion with ice-cold 250 mM sucrose solution, the livers were immediately removed and homogenized with 10 mM Tris᎐HCl buffer ŽpH 7.5. containing 250 mM sucrose and 10 mM EDTA, using a Potter᎐Elvehjem homogenizer. All subsequent procedures were performed at 3᎐5⬚C. The homogenate was centrifuged at 600 = g for 10 min to sediment the nuclei and cell debris. The resulting supernatant was centrifuged at 9000 = g for 20 min to sediment mitochondria. The mitochondrial pellets were resuspended and then centrifuged at 15 000 = g for 5 min. Furthermore, the 9000 = g supernatant was centrifuged at 105 000 = g for 60 min to obtain the microsomal pellets and cytosolic fraction. The microsomal pellets were resuspended and then centrifuged at 105 000 = g for 60 min. The reaction mixture consisted of 100 mM potassium phosphate buffer ŽpH 7.4., 500 M cofactor ŽNADPH or NADH., substrate ŽNDP. at various concentrations, and enzyme preparation Žmitochondria, cytosol, microsomes, or mixture of mitochondria and cytosol., in a total volume of 2.0 ml. After incubation at 37⬚C for 15 min, aliquots Ž1.0 ml. of the reaction mixtures were taken and PGL formed was determined by HPLC as described below.
2.4. Preparation of hepatocytes and incubation
2.6. HPLC analysis
Animals were killed by decapitation. Hepatocytes were carefully prepared from rat livers according to the method of Moldeus ´ et al. Ž1978.. The cell concentration of the hepatocytes was adjusted to 8 = 10 6 cellsrml. Incubation was carried out in Krebs᎐Henseleit buffer ŽpH 7.4. con-
To 1.0 ml of the incubated samples, 0.1 N Na 2 CO 3 Ž0.3 ml. and methanol Ž0.1 ml. containing Tetra-P and Tri-Q as the internal standard were added, and the mixture was extracted with 3.0 ml of chloroform. The organic layer was removed and evaporated to dryness. The residue
2.3. Animals
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was dissolved in methanol Ž0.1 ml. and subjected to HPLC for the determination of NDP, AcNDP and PGL. The aqueous layer was acidified by adding 2 N HCl Ž1.0 ml. containing ETP as the internal standard and extracted with 4.0 ml of ethyl acetate. The organic layer was removed and evaporated to dryness. The residue was dissolved in methanol Ž0.1 ml. and subjected to HPLC for the determination of NLA and NAA. HPLC was carried out using a Shimadzu LC-10A apparatus equipped with a reversed phase column ŽTSK gel ODS-80 TM, 25 cm = 4.6 mm, Tosoh, Tokyo, Japan . and a spectrofluorometric detector ŽShimadzu RF-550.. The column eluate was monitored at 310 nm Žexcitation. and 380 nm Žemission.. The column temperature was 37⬚C and the flow rate was 0.8 mlrmin. The mobile phase was a mixture of acetonitrilermethanolrwaterracetic acid Ž18:7:75:1.. 2.7. Determination of protein content Protein content was determined by using the BIO-RAD protein assay kit ŽBIO-RAD, Hercules, CA.. 2.8. Statistical analysis Data were expressed as the means " S.D. of three experiments. The statistical significance of differences was analyzed by Student’s t-test, and P- 0.05 was considered significant.
3. Results 3.1. Metabolism of NDP in isolated rat hepatocytes The further metabolism of NDP, a side-chain metabolite of PL, was examined in isolated rat hepatocytes. As shown in Fig. 2a, a rapid disappearance of NDP from the incubation medium was observed. Furthermore, two major metabolites, NLA and PGL, were generated from NDP, accompanied by small amounts of NAA and AcNDP ŽFig. 2b.. However, there was a large difference between the amounts of NDP remaining and these four metabolites formed Žsee Fig. 2a,b., indicating the formation of metabolites other than NLA, PGL, NAA and AcNDP. We also examined the effect of NTE, which is a potent inhibitor of MAO found by us ŽKai et al.,
Fig. 2. Disappearance of NDP from the incubation medium of isolated rat hepatocyte Ža. and formation of various metabolites from NDP in isolated rat hepatocyte Žb.. NDP Ž500 M. was used as the substrate. Each point represents the mean " S.D. of three experiments. Žb. ', NLA; I, PGL; 䢇, NAA; ^, AcNDP.
1985., on the disappearance of NDP from the incubation medium. The inhibitory potency against MAO of NTE is known to be comparable to those of typical MAO inhibitors, iproniazid and nialamide ŽKai et al., 1985, 1988.. As expected, NTE significantly retarded the disappearance of NDP from the incubation medium ŽFig. 3.. This finding suggests that NTE decreases the formation of the aldehyde intermediate from NDP by inhibiting MAO. 3.2. Metabolism of NDP in mixture of rat li¨ er mitochondria and cytosol with NADPH Fig. 4 shows the effects of phenobarbital and
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Fig. 3. Effect of NTE on the disappearance of NDP from the incubation medium of isolated rat hepatocytes. The concentrations of NDP and NTE used as the substrate and inhibitor, respectively, were 100 M. Each point represents the mean " S.D. of three experiments. I, control; `, in the presence of NTE. U P- 0.05, UUU P- 0.001, significantly different from control value in the corresponding time.
4-NBA on the formation of PGL from NDP in a mixture of rat liver mitochondria and cytosol with NADPH Žmitochondria q cytosol with NADPH.. Phenobarbital and 4-NBA were used as a specific inhibitor of aldehyde reductase ŽTurner and Hick, 1976; Felsted et al., 1977; Ansari et al., 1991. and as a substrate inhibitor because of a typical substrate of aldehyde reductase ŽDaly and Mantle, 1982; Ellis and Hayes, 1995., respectively. These two compounds significantly decreased the PGL formation. Furthermore, the effect of 4-NBA on the PGL formation was kinetically analyzed. As evident from the Dixon-plots, 4-NBA was a competitive inhibitor of the enzyme responsible for the PGL formation ŽFig. 5.; 4-NBA was confirmed to be reduced to 4-nitrobenzyl alcohol in rat liver cytosol with NADPH Ždata not shown.. The pH dependency for the formation of PGL from NDP was also examined in the reaction mixture. As shown in Fig. 6, the pH optimum of the PGL formation was observed at approximately 8.0.
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Fig. 4. Effects of phenobarbital ŽPB. and 4-NBA on the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH. NDP Ž100 M. was used as the substrate. Each bar, expressed as the percentage of control, represents the mean " S.D. of three experiments. UU P - 0.01, UUU P - 0.001, significantly different from control value.
indicating the enantioselective metabolism of NDP to PGL.
4. Discussion The present study demonstrated that PGL is generated from NDP as a major metabolite in isolated rat hepatocytes. The metabolic reaction of NDP to PGL probably proceeds through an aldehyde intermediate generated from NDP by MAO. This was supported from the fact that the
3.3. Enantioselecti¨ e metabolism of NDP in isolated rat hepatocytes The metabolism of R-NDP and S-NDP to PGL was compared in isolated rat hepatocytes. The amount of PGL formed from S-NDP was larger than that of PGL formed from R-NDP ŽFig. 7.,
Fig. 5. Dixon-plots for the inhibition by 4-NBA of PGL formation in the mixture of rat liver mitochondria and cytosol with NADPH. Velocity Ž V . is expressed as nmolrminrmg protein. The concentrations of NDP used as the substrate were 10 M ŽB., 20 M Ž䢇. and 40 M Ž'..
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Fig. 6. pH dependency for the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH. NDP Ž100 M. was used as the substrate. Each point represents the mean " S.D. of three experiments.
PGL formation is decreased by adding NTE, a potent inhibitor of MAO, to the incubation medium. To establish the characteristics of enzymes responsible for the metabolism of NDP to PGL in rat liver, a preliminary study was carried out by using subcellular fractions Žmitochondria, cytosol, microsomes and their mixtures. prepared from rat liver Ždata not shown.. PGL was most efficiently formed from NDP in a mixture of mitochondria and cytosol with NADPH. On the other hand, PGL was little formed from NDP when NADPH was replaced by NADH, and in
Fig. 7. Formation of PGL from R-NDP or S-NDP in isolated rat hepatocytes. R-NDP Ž100 M. or S-NDP Ž100 M. was used as the substrate. Each point represents the mean " S.D. of three experiments. `, from R-NDP; I, from S-NDP. UU P- 0.01, UUU P- 0.001, significantly different from the amount of PGL formed from R-NDP in the corresponding time.
the enzyme reaction system of mitochondria alone, microsomes alone or cytosol with NADPH. These results lead us to suggest that in addition to MAO Žmitochondrial enzyme., an NADPHdependent cytosolic enzyme is involved in the metabolism of NDP to PGL in rat liver. In this study, we revealed that phenobarbital and 4-NBA significantly decrease the formation of PGL from NDP in the mixture of rat liver mitochondria and cytosol with NADPH, and that 4-NBA is a competitive inhibitor of the enzyme responsible for the PGL formation, based on the kinetic data of Dixon-plots. Phenobarbital is a specific inhibitor of aldehyde reductase ŽTurner and Hick, 1976; Felsted et al., 1977; Ansari et al., 1991. and 4-NBA is widely used as a substrate of aldehyde reductase ŽDaly and Mantle, 1982; Ellis and Hayes, 1995.. Thus, it is reasonable to assume that the metabolic reduction of the aldehyde intermediate to PGL is catalyzed by aldehyde reductase. More recently, it has been reported that aldose reductase, which is a cytosolic enzyme like aldehyde reductase, mainly contributes to the metabolism of norepinephrine to 3,4-dihydroxyphenylglycol in the presence of MAO ŽKawamura et al., 1999.. Aldose reductase and carbonyl reductase also have the ability to reduce aldehydes. However, aldose reductase and carbonyl reductase are not inhibited by phenobarbital ŽAhmed et al., 1981; Ansari et al., 1991., suggesting that aldehyde reductase rather than these two enzymes is mainly involved in the metabolic reduction of the aldehyde intermediate to PGL. Aldehyde reductase purified from rat liver has an optimal pH of 8.5 when daunorubicin, an effective anticancer drug, is used as a substrate ŽFelsted et al., 1977.. A similar pH optimum is observed for daunorubicin reduction catalyzed by rabbit liver aldehyde reductase ŽAhmed et al., 1981.. On the other hand, aldose reductase and carbonyl reductase have their maximum activity in the range of pH 6.0᎐6.5 ŽPropper and Maser, ¨ 1997.. In this study, the optimal pH for the formation of PGL from NDP was approximately 8.0 in the reaction mixture of rat liver mitochondria and cytosol with NADPH. This result also supports the idea that the metabolism of the aldehyde intermediate to PGL is mainly mediated by aldehyde reductase. So far, little attempt has been made to evaluate the contribution of aldehyde reductase to drug metabolism. This is because,
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among clinically used drugs, there is no drug having an aldehyde group within its chemical structure. However, aldehyde intermediates are known to be generated as metabolites of endogenous and exogeneous compounds including drugs. It is possible that aldehyde intermediates are highly reactive molecules and induce a variety of toxic effects. We propose the importance of aldehyde reductase as one of drug-metabolizing enzymes. Fujita et al. Ž1993. have shown the enantioselectivity for the oxidation Ž4-, 5- and 7-hydroxylations and N-desisopropylation. of propranolol by CYPs purified from rat liver. Species difference in the enantioselective oxidation of propranolol is also examined ŽNarimatsu et al., 2000.. However, enantioselectivity for the further metabolism of NDP generated by the N-desisopropylation of propranolol remains to be elucidated. We found that in isolated rat hepatocytes, the amount of PGL formed from S-NDP is larger than that of PGL formed from R-NDP. Either MAO or aldehyde reductase may play a critical role in the enantioselective metabolism of NDP to PGL. Further studies are in progress to establish the enzymology for the enantioselective metabolism of NDP to PGL in rat liver. In conclusion, the data obtained in the present study demonstrate that in the rat liver, MAO catalyzes the oxidative deamination of NDP to an aldehyde intermediate and the formed aldehyde intermediate is subsequently reduced to PGL by aldehyde reductase. References Abramovitch, R.A., Takaya, T., 1972. The reaction of sulfonyl azides with pyridines and fused pyridine derivatives. J. Org. Chem. 37, 2022᎐2029. Ahmed, N.K., Felsted, R.L., Bachur, N.R., 1981. Daunorubicin reduction mediated by aldehyde and ketone reductases. Xenobiotica 11, 131᎐136. Ansari, N.H., Bhatnagar, A., Liu, S., Srivastava, S.K., 1991. Purification and characterisation of aldose reductase and aldehyde reductase from human kidney. Biochem. Int. 25, 755᎐765. Bargar, E.M., Walle, U.K., Bai, S.A., Walle, T., 1983. Quantitative metabolic fate of propranolol in the dog, rat and hamster using radiotracer, high performance liquid chromatography, and gas chromatography᎐mass spectrometry techniques. Drug Metab. Dispos. 11, 266᎐272.
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Daly, A.K., Mantle, T.J., 1982. Purification and characterisation of the multiple forms of aldehyde reductase in ox kidney. Biochem. J. 205, 373᎐380. Ellis, E.M., Hayes, J.D., 1995. Substrate specificity of an aflatoxin-metabolizing aldehyde reductase. Biochem. J. 312, 535᎐541. Felsted, R.L., Richter, D.R., Bachur, N.R., 1977. Rat liver aldehyde reductase. Biochem. Pharmacol. 26, 1117᎐1124. Fujita, S., Umeda, S., Funae, Y. et al., 1993. Regio- and stereoselective propranolol metabolism by 15 forms of purified cytochrome P-450 from rat liver. J. Pharmacol. Exp. Ther. 264, 226᎐233. Haegele, K.D., Skrdlant, H.B., Robie, N.W., Lalka, D., MaNay Jr., J.L., 1976. Determination of hydralazine and its metabolites by gas chromatography-mass spectrometry. J. Chromatogr. 126, 517᎐534. Kai, M., Noda, A., Noda, H., Goto, S., 1985. Detection of a new series of monoamine oxidase inhibitors. Chem. Pharm. Bull. 33, 5585᎐5588. Kai, M., Noda, A., Noda, H., Goto, S., 1988. Structure᎐activity relationship of a new series of tricyclic monoamine oxidase inhibitors of pentanthrene type. Chem. Pharm. Bull. 36, 3604᎐3608. Kawamura, M., Eisenhofer, G., Kopin, I.J. et al., 1999. Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats. Biochem. Pharmacol. 58, 517᎐524. Masubuchi, Y., Hosokawa, S., Horie, T. et al., 1994. Cytochrome P450 isozymes involved in propranolol metabolism in human liver microsomes. The role of CYP2D6 as ring-hydroxylase and CYP1A2 as N-desisopropylase. Drug Metab. Dispos. 22, 909᎐915. Moldeus, J., Orrenius, S., 1978. Isolation ´ P., Hogberg, ¨ and use of liver cells. In: Fleisher, S., Packer, L. ŽEds.., Methods in Enzymology, 52. Academic Press, New York, pp. 60᎐71. Narimatsu, S., Kobayashi, N., Masubuchi, Y. et al., 2000. Species difference in enantioselectivity for the oxidation of propranolol by cytochrome P450 2D enzymes. Chem. Biol. Interact. 127, 73᎐90. Noda, A., Ono, Y., Inokuchi, K. et al., 1994. Detection of 1-acetamino-3-Ž1-naphthyloxy.-2-propanol as a new metabolite of propranolol. Biol. Pharm. Bull. 17, 1433᎐1435. Oppermann, U.C., Maser, E., 2000. Molecular and structural aspects of xenobiotic carbonyl metabolizing enzymes. Role of reductases and dehydrogenases in xenobiotic phase I reactions. Toxicology 144, 71᎐81. Propper, D., Maser, E., 1997. Carbonyl reduction of ¨ daunorubicin in rabbit liver and heart. Pharmacol. Toxicol. 80, 240᎐245. Routlege, P.A., Shand, D.G., 1979. Clinical pharmacokineics of propranolol. Clin. Pharmacokinet. 4, 73᎐90.
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X. Wu et al. r Comparati¨ e Biochemistry and Physiology Part C 129 (2001) 361᎐368
Turner, A.J., Hick, P.E., 1976. Metabolism of daunorubicin by a barbiturate-sensitive aldehyde reductase from rat liver. Biochem. J. 159, 819᎐822. Vasiliou, V., Pappa, A., Petersen, D.R., 2000. Role of aldehyde dehydrogenase in endogenous and xenobiotic metabolism. Chem. Biol. Interact. 129, 1᎐19. Walle, T., Gaffney, T.E., 1972. Propranolol metabolism in man and dog: mass spectrometric identification of six new metabolites. J. Pharmacol. Exp. Ther. 182, 83᎐92. Walle, T., Walle, U.K., Olanoff, L.S., 1985. Quantitative account of propranolol metabolism in urine of normal man. Drug Metab. Dispos. 13, 204᎐209. Walle, T., Webb, J.G., Bagwell, E.E., Walle, U.K.,
Daniell, H.B., Gaffney, T.E., 1988. Stereoselective delivery and action of beta receptor antagonists. Biochem. Pharmacol. 37, 115᎐124. Wermuth, B., Munch, J.D.B., 1979. Reduction of bio¨ genic aldehydes by aldehyde reductase and alcohol dehydrogenase from human liver. Biochem. Pharmacol. 28, 1431᎐1433. Yoshimoto, K., Echizen, H., Chiba, K., Tani, M., Ishizaki, T., 1995. Identification of human CYP isoforms involved in the metabolism of propranolol enantiomers. N-Desisopropylation is mediated mainly by CYP1A2. Brit. J. Clin. Pharmacol. 39, 421᎐431.