[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES
509
the relevant controls (e.g., minus generating system). Further, if expressing the effect of antibody inhibition on mutagenicity as a percentage of original activity, it is appropriate to give original activities. This ensures the reader can work backward from the transformation and accurately derive the original data. Biohazard Requirements. The wild-type Salmonella typhimurium strain can cause food poisoning. Although the genetically manipulated strains used in the Ames test are not very virulent, it is prudent to use care when handling these bacteria. 4 Since many of the xenobiotics used will be mutagenic and carcinogenic, extreme care should be taken to prevent human exposure. Workers should consult local Biohazard Committee guidelines before commencing an experiment. Ideally all laboratory materials used should be of the disposable type, and guidelines for the disposal of both biological and carcinogenic waste should be strictly followed.
[49] B u f u r a l o l , D e x t r o m e t h o r p h a n , a n d D e b r i s o q u i n e as P r o t o t y p e S u b s t r a t e s for H u m a n P 4 5 0 I I D 6
By
THOMAS
KRONBACH
Introduction The human debrisoquine/sparteine-type genetic polymorphism of drug oxidation affects the expression of cytochrome P450IID6,1,2 an enzyme which is involved in the metabolism of many drugs (reviewed in Ref. 3). Although the P450 enzymes are often believed to exhibit a broad and overlapping substrate specificity, many drugs which are substrates for P450IID6 are only marginally metabolized in individuals affected by this polymorphism (poor metabolizers). This indicates that, among the other hepatic P450s, P450IID6 has a unique substrate selectivity and is the major catalyst involved in the clearance of these drugs. This chapter describes assays for the metabolism of three prototype substrates for P450IID6. The experimental/t-blocking agent bufuralol is metabolized by P450IID6 by l'-hydroxylation, the antihypertensive drug debrisoquine is metabolized by 4-hydroxylation, and dextromethorphan is F. J. Gonzalez, R. C. Skoda, S. Kimura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 2 U. M. Zanger, F. Vilbois, J. P. Hardwick,and U. A. Meyer,Biochemistry27, 5447(1988). 3 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
510
ENZYME ASSAYS
[49]
~N Bufuralol
NH II C~NH2 Debrisoquine
H3C~I~ ~~-~O_~H 3 Dextromethorphan FIG. 1. Structures of bufuralol, debrisoquine, and dextromethorphan. The point of metabolic attack by P450IID6 is indicated by arrows,
converted to its O-demethyl metabolite dextrorphan. The chemical structures of these substances and the points of metabolic attack of P450IID6 are shown in Fig. 1. The applications of these assays are numerous and include the functional characterization of the enzyme expressed in microsomes prepared from kidney transplant donor livers 4 or from liver biopsies obtained from poor and extensive metabolizers which had been phenotyped in vivo. 5 Moreover, these assays have been used to monitor the purification of the human enzyme, 6 in the investigation of the enzyme expressed from its eDNA in COS-I cells,l and the characterization of autoantibodies in autoimmune hepatitis type II which are directed against P450IID67 (see also [20], this volume). The choice of substrate depends on the particular question. We have used (+)- and (-)-bufuralol extensively for all the above in vitro studies mostly because P450IID6 is stereoselective in that it preferentially catalyzes l'-hydroxylation of (+)-bufuralol and the loss of stereoselectivity characterizes the carriers of the genetic deficiency 4 T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24
(1987). 5 p. Dayer, R. Gasser, J. Gut, T. Kronbach, G.-M. Robertz, M. Eichelbaum, and U. A. Meyer, Biochem. Biophys. Res. Commun. 125, 374 (1984). 6 j. Gut, T. Catin, P. Dayer, T. Kronbach, U. Zanger, and U. A. Meyer, J. Biol. Chem. 261, 11734 (1986). 7 U. M. Zanger, H.-P. Hauri, J. Loeper, J.-C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988).
[49]
HPLC ASSAYSFOR HUMAN P450IID6 SUBSTRATES
51 1
(poor metabolizers). 5,s'9 M o r e o v e r , l'-hydroxybufuralol is highly fluorescent, which enables sensitive and selective detection yielding a highly reproducible system. Interestingly, P450IID6 can also use cumene hydroperoxide as an oxygen donor for the hydroxylation of debrisoquine and bufuralol with an apparent Km and stereoselectivity similar to the reaction supported by N A D P H . 2 This reaction can also be analyzed with the highperformance liquid chromatography (HPLC) method described here.
Assays All three assays are based on the same principle in that they use a nonextractive sample preparation which not only allows the quantification o f the products by external standardization but is also easy to perform. After incubation of the microsomes in the presence of substrate and a NADPH-generating system, the protein is precipitated from the suspension b y perchloric acid, and the compounds are then chromatographed after centrifugation in a perchlorate-containing eluent under isocratic conditions. The perchlorate ions presumably form ion pairs with the basic nitrogen c o m m o n to all substrates for P450IID6,1° which results in sharp, symmetrical peaks and preserves the column. It is interesting to note that this eluent system also yields good resolution for the separation of other basic drugs and their metabolites such as N-alkyl-substituted 4,4-diphenylpiperidines, H propafenone, 12 or sulfamethazine. 13 The assays described here derive their sensitivity from the fluorescence detection which is employed. This is especially advantageous when the amount of material is limited, as is normally the case with liver biopsy samples, or when the specific content of the sample is low, for example, microsomal fractions prepared from transfected cells. The specificity of fluorescence detection can be exploited in that a vast number o f inhibitors and/or potential substrates such as quinidine, ~4 propafenone, z2 mephenytoin, ~5 and antibodies 2 can be included in the incubation mixture without interfering with the assay. 8 p. Dayer, T. Leemann, A. K0pfer, T. Kronbach, and U. A. Meyer, Eur. J. Clin. Pharmacol. 31, 313 (1986). 9 p. Dayer, T. Leemann, J. Gut, T. Kronbach, A. Kiipfer, R. Francis, and U. A. Meyer, Biochem. Biophys. Res. Commun. 34, 399 (1990). 10U. A. Meyer, J. Gut, T. Kronbach, C. Skoda, U. T. Meier, and T. Catin, Xenobiotica 16, 449 (1986). ~ W. Voelter and T. Kronbach, J. Chromatogr. 290, 1 (1984). 12H. K. Kroerner, G. Mikus, T. Kronbach, U. A. Meyer, and M. Eichelbanm, Clin. Pharmacol. Ther. 45, 28 (1989). 13D. M. Grant, F. Lottspeich, and U. A. Meyer, FEBS Lett. 244, 203 (1989). 14p. Dayer, T. Kronbach, M. Eichlebaum, and U. A. Meyer, Biochem. Pharmacol. 36, 4145 (1987). 15U. T. Meier, T. Kronbach, and U. A. Meyer, Anal. Biochem. 151, 286 (1985).
512
[49]
ENZYME ASSAYS TABLE I CONDITIONS FOR MICROSOMALINCUaATIONS AND HPLC ASSAYSFOR I'-HYDROXYLATION OF BUFURALOL, 4-HYDROXYLATION OF DEIIRISOQUINE, AND O-DEMETHYLATION OF DEXTROMETHORPHANa
Parameter Microsomal protein (/zg) Injection volume (/zl) Flow rate (ml/min) Volume % acetonitrile in
Bufuralol
Debrisoquine
Dextromethorphan
10 10 2.2 45
50 100 1.0 15
20 50 1.0 27
Shperisorb ODS
Nucleosil Cl8
Nucleosil C~8
219 286 99.8 - 1.4 60 2.9
270 312 103.7 -+ 1.7 45 0.4
20 mM NaCIO4, pH 2.5
Column filling material (particle size 5/zm) Fluorescence detection Excitation (nm) Emission (nm) Metabolite recovery (%) Linearity with time (min) Sensitivity (pmol/ml) at signalnoise ratio of I : 4
252 302 98.1 --- 1.8 60 0.4
a The assays are described in more detail in the text.
Materials and Equipment (+)-, ( - ) - , and (---)-bufuralol hydrochloride, debrisoquine, dextromethorphan hydrobromide, and their respective metabolites were kindly provided by Hoffman-La Roche (Basel, Switzerland). Solvents are delivered with Model 414 or 420 pumps (Kontron, Zurich, Switzerland). The choice of pump is not critical, and any modern constant flow pump is expected to work with these assays. To avoid precipitation of salt in the HPLC system we pump the eluent at 50/zl/min when the system is not in use. The flow rates for the assays are indicated in Table I. The sample is injected with an autosampler (ISS 100, Perkin-Elmer, Ktisnacht, Switzerland) or by manual injection with a loop valve (Model 7125, Rheodyne, Berkeley, CA). The method of injection is not important; however, the accuracy of the results depends largely on the accuracy of the injection when external standardization is applied, and an automated system may work more reliably if calibrated properly. The preparation of microsomes by differential centrifugation from kidney transplant donor livers 2'16 or from wedge biopsies 5,14 is described elsewhere. A variation of the latter method can also be used to prepare 16 p. j. Meier, H. K. Mueller, B. Dick, and U. A. Meyer,
Gastroenterology 85, 682 (1983).
[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES
513
microsomal fractions from transfected COS-1 cells.17 The purification of P450bufI and P450buflI are described elsewhere. 6 P450bufI was later shown to correspond to P450IID6.
Incubation of Microsomes Incubations can be performed in either glass or polypropylene tubes, but polystyrene tubes should be avoided since they absorb considerable amounts of substrate and metabolite. The conditions for microsomal incubations for a final volume of 150/.d are shown in Table I. The amount of microsomal protein indicated in Table I is diluted in 0. I M sodium phosphate buffer, pH 7.4, and is incubated at 37° for about 5 min with a NADPH-generating system. This system consists of 1 mM disodium NADP (Boehringer, Mannheim, Germany), 5 mM MgC12,5 mM isocitrate, and 1 unit ofisocitrate dehydrogenase (Sigma, St. Louis, MO) and is added as a 5-fold concentrate. The reaction is then started by the addition of a 10-fold concentrated solution of substrate in water. Depending on the activity of the microsomal sample, the mixture is incubated for 1 to 60 min. The reaction is stopped by the addition of 15/zl of perchloric acid, 60% (Merck, Darmstadt, Germany). Protein is precipitated at 10,000 g for 4 min, and an aliquot of the reaction (Table I) is injected into the HPLC system. This sample preparation is efficient in that it eliminates protein constituents which would normally clog the analytical column and also recovers metabolites which are not apparent when alternative methods are used. 18
Separation The interaction of an ionized solute with charged groups of the silica matrix of reversed-phase supports is often the cause of tailing effects which compromise resolution, sensitivity, and accurate quantification. In suppressed-ion reversed-phase chromatography charged groups of the solute are neutralized by the adjustment of the pH. Unfortunately, the presence of a nitrogen in all P450IID6 substrates hinders the application of suppressed-ion chromatography because high pH values would be necessary and silica-based supports are not stable under these conditions. The addition of perchlorate ions to the eluent presumably leads to the 17T. Kronbach, T. M. Larabee, and E. F. Johnson, Proe. Natl. Acad. Sci. U.S.A. 86, 8262 (1989). 18 E. I. Minder, P. J. Meier, H. K. Mueller, C. Minder, and U. A. Meyer, Eur. J. Clin. Invest. 14, 184 (1984).
514
ENZYME ASSAYS A Blank
B Microsomes
24-
24'
16-
8"
~
[49]
C P450 bull
D P450 buf II
12-
24.
16-
8
16-
"" ,::, -,-:-
8-
4.
8"
i
O-
O.
8 " ,=, 0
"-
®
¢,,,-
O,
,
,
0 2 4
"
,
6min
~ ,
0
2 ,4 6min
,
0•
,
v
,
,
0 2 4 6min 0 2 4
,
6rain
FIG. 2. Chromatograms of the metabolites of (+)-bufuralol formed by the incubation of microsomes and enzyme fractions prepared from human liver. (A) Microsomal incubation without substrate. (B) Microsomal incubation with (+)-bufuralol; I'-OH marks V-OHbufuralol, and M 1, M2, and M3 refer to metabolites formed by isozymes other than P450IID6. (C) l'-OH-bufuralol formed by purified P450IID6 (P450bufI). (D) Metabolites formed by a purified preparation of P450buflI, an isozyme for which the sequence has not yet been reported. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.]
formation of tight ion pairs. Such ion pairs can sometimes be extracted into organic solvents, which demonstrates that the charge of the organic ion is efficiently masked. The eluent used here takes advantage of this effect, and the chaotropic properties of perchloric acid are used to precipitate the protein. In addition, the perchloric acid also forms the ion pairs prior to injection. The eluent consists of 20 mM sodium perchlorate (Merck) in HPLCgrade water and is adjusted to a pH of 2.5 with perchloric acid (60%). This solution is then filtered through a 4.5-/~m filter, and acetonitrile is added to the amount indicated in Table I. When a new batch of column material is used, the percentage of acetonitrile is adjusted so that l'-hydroxybufuralol elutes at approximately 2.1 to 2.4 rain and the metabolites of debrisoquine and dextromethorphan elute between 4 and 6 min (Figs. 2, 3, and 4). Stainless steel columns (4.6 x 125 mm, Bischoff Analysentechnick, Leonberg, Germany) were packed with either Sherisorb ODS (dp 5/zm, Phase Separations, Norwalk, CT) or Nucleosil 5-C1s (dp 5/xm, MacheryNagel, Dfiren, Germany) by means of an imbalanced slurry technique at 550 bars. The Sherisorb ODS columns were used for the assay of bufuralol metabolism because they provided the best selectivity, and the Nucleosil Cls columns were used for the analysis of the metabolism of debrisoquine
[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES B Microsomes
A Blank
C P450 bufl
515 D P450 buf II
16
12 A
,4-
E ,=, ==
==
=.= =D ee,"
4-
,
'
0
v
•
2
4
,
6rain
v
i
i
0
2
4
i
6rain
,
i
v
0
2
4
i
6min
f
,
1
0
2
4
i
6rain
FzG. 3. Chromatograms of debrisoquine metabolites formed in a microsomal incubation without (A) or with (B) substrate and formed by P450IID6 (P450bufI) (C) and P450buflI (D). 6-OH and 4-OH refer to 6- and 4-hydroxydebrisoquine, respectively. MI designates an unknown metabolite of debrisoquine. The peaks eluting after MI are impurities present in the substrate. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.]
and dextromethorphan. Commercially available columns which were packed with the same materials gave essentially the same selectivity. Detection Detection was performed with 650 and 650S fluorescence detectors (Perkin-Elmer) with slit widths set at 20 nm for both, emission and excitation. These detectors use high-pressure xenon lamps as the light source, which provides excellent energy throughout above 220 nm. For lower excitation wavelengths, which can also be used for the detection of dextromethorphan (excitation 198 nm, emission 330 nm) with a sensitivity of approximately 70 pmol/ml, 19 a detector employing a deuterium lamp as light source should be used. I9 p. Dayer, T. Leemann, and R. Striberni, Clin. Pharmacol. Ther. 45, 34 (1989).
516
ENZYME ASSAYS 256-
A Blank
B Microsomes
[49]
C P450 bufl
D P450 buf II
---- O-Deme 192c
L,.
~ 128 ',~
ll+
"" 64
0
N
J
..,.a,_
2 4 6rain
0 2 4 6min
Ft6.4. Chromatogramof the metabolitesof dextromethorphanformedby the incubation of human liver microsomes without (A) or with (B) substrate and formed by P450IID6 (P450bufl) (C) and P450buflI(D). O-Demerefers to the O-demethylmetabolitedextrorphan. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.] Quantification
Fluorescence detection can yield very high sensitivity which compares favorably with radiometric methods. However, in contrast to radiometric methods where scintillation counting yields similar sensitivity for compound and metabolites, accurate quantification of the compounds of interest by fluorescence detection may become difficult. Although it is common practice for detection systems based on the absorption of ultraviolet light to assume, in a rough approximation, that the extinction coefficients of parent compound and metabolite are identical, it cannot be assumed that metabolites fluorescence at similar wavelengths and with similar intensities as the parent compound. This is due to the influence of newly introduced functional groups in the metabolite on the fluorophore which may lead to a dramatic alteration of the fluorescence parameters. For example, l'-ketobufuralol is a metabolite of the highly fluorescent bufuralol which does not fluorescence at all and which is, therefore, not detected by the assay described here. Thus, it is mandatory to use the authentic metabolites, l'-hydroxybufuralol for bufuralol, 4-hydroxydebrisoquine for debrisoquine, and dextrorphan for dextromethorphan to establish a calibration curve.
[50]
C O 2 B R E A T H TESTS
517
Signals from the fluorescence detectors were integrated with C-R3A integrators (Shimadzu, Kyoto, Japan), but any modem integrator should perform satisfactorily, provided it can be adapted to a wide range of slope sensitivities and peak widths. This is especially important when the maximum of excitation and emission are close together. For example, under the conditions used for dextromethorphan, the excitation and emission are 42 nm apart, and the slit width for both wavelengths reduces this difference to only 22 nm. This yields a baseline where the noise is composed of very sharp spikes which are sometimes difficult to distinguish from true peaks. Acknowledgments This work was performed at the Department of Pharmacology at the Biocenter of the University, Basel, Switzerland, and was supported by the Swiss National Science Foundation.
[50] B r e a t h T e s t s as N o n i n v a s i v e Assays of P 4 5 0 s By PAUL B. WATKINS Introduction Formaldehyde is the immediate product of P450-catalyzed demethylation reactions. A large proportion of the formaldehyde rapidly undergoes sequential conversions to formate and bicarbonate by enzymes that are believed to be ubiquitous in the body. As a result, approximately one-half of the carbon atoms derived from formaldehyde promptly appears in the breath as carbon dioxide. Thus, the rate of production of breath CO2 from a suitable substrate may reflect the in vivo rate of its demethylation, which may in turn reflect the catalytic activity of a subset of P450s. The major advantage of breath tests is that they are extremely easy to perform. By measuring the rate of a single substrate reaction (demethylation), breath tests also have a theoretical advantage over measurements of blood clearance when dealing with substrates that have multiple metabolic pathways. Breath tests have many potential pitfalls, however. First, the test relies on the assumption that the P450-catalyzed formation of formaldehyde is rate limiting in the entire cascade of steps that result in the formation of breath CO2. These steps include delivery of the substrate to and uptake of the substrate by the liver, transport of the substrate to the endoplasmic reticulum, and the subsequent conversion of the formaldeMETHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc, All rights of reproduction in any form reserved.
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES
509
the relevant controls (e.g., minus generating system). Further, if expressing the effect of antibody inhibition on mutagenicity as a percentage of original activity, it is appropriate to give original activities. This ensures the reader can work backward from the transformation and accurately derive the original data. Biohazard Requirements. The wild-type Salmonella typhimurium strain can cause food poisoning. Although the genetically manipulated strains used in the Ames test are not very virulent, it is prudent to use care when handling these bacteria. 4 Since many of the xenobiotics used will be mutagenic and carcinogenic, extreme care should be taken to prevent human exposure. Workers should consult local Biohazard Committee guidelines before commencing an experiment. Ideally all laboratory materials used should be of the disposable type, and guidelines for the disposal of both biological and carcinogenic waste should be strictly followed.
[49] B u f u r a l o l , D e x t r o m e t h o r p h a n , a n d D e b r i s o q u i n e as P r o t o t y p e S u b s t r a t e s for H u m a n P 4 5 0 I I D 6
By
THOMAS
KRONBACH
Introduction The human debrisoquine/sparteine-type genetic polymorphism of drug oxidation affects the expression of cytochrome P450IID6,1,2 an enzyme which is involved in the metabolism of many drugs (reviewed in Ref. 3). Although the P450 enzymes are often believed to exhibit a broad and overlapping substrate specificity, many drugs which are substrates for P450IID6 are only marginally metabolized in individuals affected by this polymorphism (poor metabolizers). This indicates that, among the other hepatic P450s, P450IID6 has a unique substrate selectivity and is the major catalyst involved in the clearance of these drugs. This chapter describes assays for the metabolism of three prototype substrates for P450IID6. The experimental/t-blocking agent bufuralol is metabolized by P450IID6 by l'-hydroxylation, the antihypertensive drug debrisoquine is metabolized by 4-hydroxylation, and dextromethorphan is F. J. Gonzalez, R. C. Skoda, S. Kimura, M. Umeno, U. M. Zanger, D. W. Nebert, H. V. Gelboin, J. P. Hardwick, and U. A. Meyer, Nature (London) 331, 442 (1988). 2 U. M. Zanger, F. Vilbois, J. P. Hardwick,and U. A. Meyer,Biochemistry27, 5447(1988). 3 U. A. Meyer, R. C. Skoda, and U. M. Zanger, Pharmacol. Ther. 46, 297 (1990). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
510
ENZYME ASSAYS
[49]
~N Bufuralol
NH II C~NH2 Debrisoquine
H3C~I~ ~~-~O_~H 3 Dextromethorphan FIG. 1. Structures of bufuralol, debrisoquine, and dextromethorphan. The point of metabolic attack by P450IID6 is indicated by arrows,
converted to its O-demethyl metabolite dextrorphan. The chemical structures of these substances and the points of metabolic attack of P450IID6 are shown in Fig. 1. The applications of these assays are numerous and include the functional characterization of the enzyme expressed in microsomes prepared from kidney transplant donor livers 4 or from liver biopsies obtained from poor and extensive metabolizers which had been phenotyped in vivo. 5 Moreover, these assays have been used to monitor the purification of the human enzyme, 6 in the investigation of the enzyme expressed from its eDNA in COS-I cells,l and the characterization of autoantibodies in autoimmune hepatitis type II which are directed against P450IID67 (see also [20], this volume). The choice of substrate depends on the particular question. We have used (+)- and (-)-bufuralol extensively for all the above in vitro studies mostly because P450IID6 is stereoselective in that it preferentially catalyzes l'-hydroxylation of (+)-bufuralol and the loss of stereoselectivity characterizes the carriers of the genetic deficiency 4 T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24
(1987). 5 p. Dayer, R. Gasser, J. Gut, T. Kronbach, G.-M. Robertz, M. Eichelbaum, and U. A. Meyer, Biochem. Biophys. Res. Commun. 125, 374 (1984). 6 j. Gut, T. Catin, P. Dayer, T. Kronbach, U. Zanger, and U. A. Meyer, J. Biol. Chem. 261, 11734 (1986). 7 U. M. Zanger, H.-P. Hauri, J. Loeper, J.-C. Homberg, and U. A. Meyer, Proc. Natl. Acad. Sci. U.S.A. 85, 8256 (1988).
[49]
HPLC ASSAYSFOR HUMAN P450IID6 SUBSTRATES
51 1
(poor metabolizers). 5,s'9 M o r e o v e r , l'-hydroxybufuralol is highly fluorescent, which enables sensitive and selective detection yielding a highly reproducible system. Interestingly, P450IID6 can also use cumene hydroperoxide as an oxygen donor for the hydroxylation of debrisoquine and bufuralol with an apparent Km and stereoselectivity similar to the reaction supported by N A D P H . 2 This reaction can also be analyzed with the highperformance liquid chromatography (HPLC) method described here.
Assays All three assays are based on the same principle in that they use a nonextractive sample preparation which not only allows the quantification o f the products by external standardization but is also easy to perform. After incubation of the microsomes in the presence of substrate and a NADPH-generating system, the protein is precipitated from the suspension b y perchloric acid, and the compounds are then chromatographed after centrifugation in a perchlorate-containing eluent under isocratic conditions. The perchlorate ions presumably form ion pairs with the basic nitrogen c o m m o n to all substrates for P450IID6,1° which results in sharp, symmetrical peaks and preserves the column. It is interesting to note that this eluent system also yields good resolution for the separation of other basic drugs and their metabolites such as N-alkyl-substituted 4,4-diphenylpiperidines, H propafenone, 12 or sulfamethazine. 13 The assays described here derive their sensitivity from the fluorescence detection which is employed. This is especially advantageous when the amount of material is limited, as is normally the case with liver biopsy samples, or when the specific content of the sample is low, for example, microsomal fractions prepared from transfected cells. The specificity of fluorescence detection can be exploited in that a vast number o f inhibitors and/or potential substrates such as quinidine, ~4 propafenone, z2 mephenytoin, ~5 and antibodies 2 can be included in the incubation mixture without interfering with the assay. 8 p. Dayer, T. Leemann, A. K0pfer, T. Kronbach, and U. A. Meyer, Eur. J. Clin. Pharmacol. 31, 313 (1986). 9 p. Dayer, T. Leemann, J. Gut, T. Kronbach, A. Kiipfer, R. Francis, and U. A. Meyer, Biochem. Biophys. Res. Commun. 34, 399 (1990). 10U. A. Meyer, J. Gut, T. Kronbach, C. Skoda, U. T. Meier, and T. Catin, Xenobiotica 16, 449 (1986). ~ W. Voelter and T. Kronbach, J. Chromatogr. 290, 1 (1984). 12H. K. Kroerner, G. Mikus, T. Kronbach, U. A. Meyer, and M. Eichelbanm, Clin. Pharmacol. Ther. 45, 28 (1989). 13D. M. Grant, F. Lottspeich, and U. A. Meyer, FEBS Lett. 244, 203 (1989). 14p. Dayer, T. Kronbach, M. Eichlebaum, and U. A. Meyer, Biochem. Pharmacol. 36, 4145 (1987). 15U. T. Meier, T. Kronbach, and U. A. Meyer, Anal. Biochem. 151, 286 (1985).
512
[49]
ENZYME ASSAYS TABLE I CONDITIONS FOR MICROSOMALINCUaATIONS AND HPLC ASSAYSFOR I'-HYDROXYLATION OF BUFURALOL, 4-HYDROXYLATION OF DEIIRISOQUINE, AND O-DEMETHYLATION OF DEXTROMETHORPHANa
Parameter Microsomal protein (/zg) Injection volume (/zl) Flow rate (ml/min) Volume % acetonitrile in
Bufuralol
Debrisoquine
Dextromethorphan
10 10 2.2 45
50 100 1.0 15
20 50 1.0 27
Shperisorb ODS
Nucleosil Cl8
Nucleosil C~8
219 286 99.8 - 1.4 60 2.9
270 312 103.7 -+ 1.7 45 0.4
20 mM NaCIO4, pH 2.5
Column filling material (particle size 5/zm) Fluorescence detection Excitation (nm) Emission (nm) Metabolite recovery (%) Linearity with time (min) Sensitivity (pmol/ml) at signalnoise ratio of I : 4
252 302 98.1 --- 1.8 60 0.4
a The assays are described in more detail in the text.
Materials and Equipment (+)-, ( - ) - , and (---)-bufuralol hydrochloride, debrisoquine, dextromethorphan hydrobromide, and their respective metabolites were kindly provided by Hoffman-La Roche (Basel, Switzerland). Solvents are delivered with Model 414 or 420 pumps (Kontron, Zurich, Switzerland). The choice of pump is not critical, and any modern constant flow pump is expected to work with these assays. To avoid precipitation of salt in the HPLC system we pump the eluent at 50/zl/min when the system is not in use. The flow rates for the assays are indicated in Table I. The sample is injected with an autosampler (ISS 100, Perkin-Elmer, Ktisnacht, Switzerland) or by manual injection with a loop valve (Model 7125, Rheodyne, Berkeley, CA). The method of injection is not important; however, the accuracy of the results depends largely on the accuracy of the injection when external standardization is applied, and an automated system may work more reliably if calibrated properly. The preparation of microsomes by differential centrifugation from kidney transplant donor livers 2'16 or from wedge biopsies 5,14 is described elsewhere. A variation of the latter method can also be used to prepare 16 p. j. Meier, H. K. Mueller, B. Dick, and U. A. Meyer,
Gastroenterology 85, 682 (1983).
[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES
513
microsomal fractions from transfected COS-1 cells.17 The purification of P450bufI and P450buflI are described elsewhere. 6 P450bufI was later shown to correspond to P450IID6.
Incubation of Microsomes Incubations can be performed in either glass or polypropylene tubes, but polystyrene tubes should be avoided since they absorb considerable amounts of substrate and metabolite. The conditions for microsomal incubations for a final volume of 150/.d are shown in Table I. The amount of microsomal protein indicated in Table I is diluted in 0. I M sodium phosphate buffer, pH 7.4, and is incubated at 37° for about 5 min with a NADPH-generating system. This system consists of 1 mM disodium NADP (Boehringer, Mannheim, Germany), 5 mM MgC12,5 mM isocitrate, and 1 unit ofisocitrate dehydrogenase (Sigma, St. Louis, MO) and is added as a 5-fold concentrate. The reaction is then started by the addition of a 10-fold concentrated solution of substrate in water. Depending on the activity of the microsomal sample, the mixture is incubated for 1 to 60 min. The reaction is stopped by the addition of 15/zl of perchloric acid, 60% (Merck, Darmstadt, Germany). Protein is precipitated at 10,000 g for 4 min, and an aliquot of the reaction (Table I) is injected into the HPLC system. This sample preparation is efficient in that it eliminates protein constituents which would normally clog the analytical column and also recovers metabolites which are not apparent when alternative methods are used. 18
Separation The interaction of an ionized solute with charged groups of the silica matrix of reversed-phase supports is often the cause of tailing effects which compromise resolution, sensitivity, and accurate quantification. In suppressed-ion reversed-phase chromatography charged groups of the solute are neutralized by the adjustment of the pH. Unfortunately, the presence of a nitrogen in all P450IID6 substrates hinders the application of suppressed-ion chromatography because high pH values would be necessary and silica-based supports are not stable under these conditions. The addition of perchlorate ions to the eluent presumably leads to the 17T. Kronbach, T. M. Larabee, and E. F. Johnson, Proe. Natl. Acad. Sci. U.S.A. 86, 8262 (1989). 18 E. I. Minder, P. J. Meier, H. K. Mueller, C. Minder, and U. A. Meyer, Eur. J. Clin. Invest. 14, 184 (1984).
514
ENZYME ASSAYS A Blank
B Microsomes
24-
24'
16-
8"
~
[49]
C P450 bull
D P450 buf II
12-
24.
16-
8
16-
"" ,::, -,-:-
8-
4.
8"
i
O-
O.
8 " ,=, 0
"-
®
¢,,,-
O,
,
,
0 2 4
"
,
6min
~ ,
0
2 ,4 6min
,
0•
,
v
,
,
0 2 4 6min 0 2 4
,
6rain
FIG. 2. Chromatograms of the metabolites of (+)-bufuralol formed by the incubation of microsomes and enzyme fractions prepared from human liver. (A) Microsomal incubation without substrate. (B) Microsomal incubation with (+)-bufuralol; I'-OH marks V-OHbufuralol, and M 1, M2, and M3 refer to metabolites formed by isozymes other than P450IID6. (C) l'-OH-bufuralol formed by purified P450IID6 (P450bufI). (D) Metabolites formed by a purified preparation of P450buflI, an isozyme for which the sequence has not yet been reported. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.]
formation of tight ion pairs. Such ion pairs can sometimes be extracted into organic solvents, which demonstrates that the charge of the organic ion is efficiently masked. The eluent used here takes advantage of this effect, and the chaotropic properties of perchloric acid are used to precipitate the protein. In addition, the perchloric acid also forms the ion pairs prior to injection. The eluent consists of 20 mM sodium perchlorate (Merck) in HPLCgrade water and is adjusted to a pH of 2.5 with perchloric acid (60%). This solution is then filtered through a 4.5-/~m filter, and acetonitrile is added to the amount indicated in Table I. When a new batch of column material is used, the percentage of acetonitrile is adjusted so that l'-hydroxybufuralol elutes at approximately 2.1 to 2.4 rain and the metabolites of debrisoquine and dextromethorphan elute between 4 and 6 min (Figs. 2, 3, and 4). Stainless steel columns (4.6 x 125 mm, Bischoff Analysentechnick, Leonberg, Germany) were packed with either Sherisorb ODS (dp 5/zm, Phase Separations, Norwalk, CT) or Nucleosil 5-C1s (dp 5/xm, MacheryNagel, Dfiren, Germany) by means of an imbalanced slurry technique at 550 bars. The Sherisorb ODS columns were used for the assay of bufuralol metabolism because they provided the best selectivity, and the Nucleosil Cls columns were used for the analysis of the metabolism of debrisoquine
[49]
HPLC ASSAYSFOR HUMANP450IID6 SUBSTRATES B Microsomes
A Blank
C P450 bufl
515 D P450 buf II
16
12 A
,4-
E ,=, ==
==
=.= =D ee,"
4-
,
'
0
v
•
2
4
,
6rain
v
i
i
0
2
4
i
6rain
,
i
v
0
2
4
i
6min
f
,
1
0
2
4
i
6rain
FzG. 3. Chromatograms of debrisoquine metabolites formed in a microsomal incubation without (A) or with (B) substrate and formed by P450IID6 (P450bufI) (C) and P450buflI (D). 6-OH and 4-OH refer to 6- and 4-hydroxydebrisoquine, respectively. MI designates an unknown metabolite of debrisoquine. The peaks eluting after MI are impurities present in the substrate. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.]
and dextromethorphan. Commercially available columns which were packed with the same materials gave essentially the same selectivity. Detection Detection was performed with 650 and 650S fluorescence detectors (Perkin-Elmer) with slit widths set at 20 nm for both, emission and excitation. These detectors use high-pressure xenon lamps as the light source, which provides excellent energy throughout above 220 nm. For lower excitation wavelengths, which can also be used for the detection of dextromethorphan (excitation 198 nm, emission 330 nm) with a sensitivity of approximately 70 pmol/ml, 19 a detector employing a deuterium lamp as light source should be used. I9 p. Dayer, T. Leemann, and R. Striberni, Clin. Pharmacol. Ther. 45, 34 (1989).
516
ENZYME ASSAYS 256-
A Blank
B Microsomes
[49]
C P450 bufl
D P450 buf II
---- O-Deme 192c
L,.
~ 128 ',~
ll+
"" 64
0
N
J
..,.a,_
2 4 6rain
0 2 4 6min
Ft6.4. Chromatogramof the metabolitesof dextromethorphanformedby the incubation of human liver microsomes without (A) or with (B) substrate and formed by P450IID6 (P450bufl) (C) and P450buflI(D). O-Demerefers to the O-demethylmetabolitedextrorphan. [Reproduced from T. Kronbach, D. Mathys, J. Gut, T. Catin, and U. A. Meyer, Anal. Biochem. 162, 24 (1987), with permission.] Quantification
Fluorescence detection can yield very high sensitivity which compares favorably with radiometric methods. However, in contrast to radiometric methods where scintillation counting yields similar sensitivity for compound and metabolites, accurate quantification of the compounds of interest by fluorescence detection may become difficult. Although it is common practice for detection systems based on the absorption of ultraviolet light to assume, in a rough approximation, that the extinction coefficients of parent compound and metabolite are identical, it cannot be assumed that metabolites fluorescence at similar wavelengths and with similar intensities as the parent compound. This is due to the influence of newly introduced functional groups in the metabolite on the fluorophore which may lead to a dramatic alteration of the fluorescence parameters. For example, l'-ketobufuralol is a metabolite of the highly fluorescent bufuralol which does not fluorescence at all and which is, therefore, not detected by the assay described here. Thus, it is mandatory to use the authentic metabolites, l'-hydroxybufuralol for bufuralol, 4-hydroxydebrisoquine for debrisoquine, and dextrorphan for dextromethorphan to establish a calibration curve.
[50]
C O 2 B R E A T H TESTS
517
Signals from the fluorescence detectors were integrated with C-R3A integrators (Shimadzu, Kyoto, Japan), but any modem integrator should perform satisfactorily, provided it can be adapted to a wide range of slope sensitivities and peak widths. This is especially important when the maximum of excitation and emission are close together. For example, under the conditions used for dextromethorphan, the excitation and emission are 42 nm apart, and the slit width for both wavelengths reduces this difference to only 22 nm. This yields a baseline where the noise is composed of very sharp spikes which are sometimes difficult to distinguish from true peaks. Acknowledgments This work was performed at the Department of Pharmacology at the Biocenter of the University, Basel, Switzerland, and was supported by the Swiss National Science Foundation.
[50] B r e a t h T e s t s as N o n i n v a s i v e Assays of P 4 5 0 s By PAUL B. WATKINS Introduction Formaldehyde is the immediate product of P450-catalyzed demethylation reactions. A large proportion of the formaldehyde rapidly undergoes sequential conversions to formate and bicarbonate by enzymes that are believed to be ubiquitous in the body. As a result, approximately one-half of the carbon atoms derived from formaldehyde promptly appears in the breath as carbon dioxide. Thus, the rate of production of breath CO2 from a suitable substrate may reflect the in vivo rate of its demethylation, which may in turn reflect the catalytic activity of a subset of P450s. The major advantage of breath tests is that they are extremely easy to perform. By measuring the rate of a single substrate reaction (demethylation), breath tests also have a theoretical advantage over measurements of blood clearance when dealing with substrates that have multiple metabolic pathways. Breath tests have many potential pitfalls, however. First, the test relies on the assumption that the P450-catalyzed formation of formaldehyde is rate limiting in the entire cascade of steps that result in the formation of breath CO2. These steps include delivery of the substrate to and uptake of the substrate by the liver, transport of the substrate to the endoplasmic reticulum, and the subsequent conversion of the formaldeMETHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc, All rights of reproduction in any form reserved.
