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Research ArticleArticle

Comparison between Cytochrome P450 (CYP) Content and Relative Activity Approaches to Scaling from cDNA-Expressed CYPs to Human Liver Microsomes: Ratios of Accessory Proteins as Sources of Discrepancies between the Approaches

Karthik Venkatakrishnan, Lisa L. von Moltke, Michael H. Court, Jerold S. Harmatz, Charles L. Crespi and David J. Greenblatt
Drug Metabolism and Disposition December 2000, 28 (12) 1493-1504;

Abstract

Relative activity factors (RAFs) and immunoquantified levels of cytochrome P450 (CYP) isoforms both have been proposed as scaling factors for the prediction of hepatic drug metabolism from studies using cDNA-expressed CYPs. However, a systematic comparison of the two approaches, including possible mechanisms underlying differences, is not available. In this study, RAFs determined for CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 in 12 human livers using lymphoblast-expressed enzymes were compared to immunoquantified protein levels. 2C19, 2D6, and 3A4 RAFs were similar to immunoquantified enzyme levels. In contrast, 1A2 RAFs were 5- to 20-fold higher than CYP1A2 content, and the RAF:content ratio was positively correlated with the molar ratio of NADPH:CYP oxidoreductase (OR) to CYP1A2. The OR:CYP1A2 ratio in lymphoblast microsomes was 92-fold lower than in human liver microsomes. Reconstitution experiments demonstrated a 10- to 20-fold lower activity at OR:CYP1A2 ratios similar to those in lymphoblasts, compared with those in human livers. CYP2B6-containing lymphoblast microsomes had 29- and 13-fold lower OR:CYP and cytochromeb5:CYP ratios, respectively, than did liver microsomes and yielded RAFs that were 6-fold higher than CYP2B6 content. Use of metabolic rates from cDNA-expressed CYPs containing nonphysiologic concentrations of electron-transfer proteins (relative to human liver microsomes) in conjunction with hepatic CYP contents may lead to incorrect predictions of liver microsomal rates and relative contributions of individual isoforms. Scaling factors used in bridging the gap between expression systems and liver microsomes should not only incorporate relative hepatic abundance of individual CYPs but also account for differences in activity per unit enzyme in the two systems.

The cloning and heterologous expression of the drug-metabolizing human CYPs1has resulted in the commercial availability of cDNA-expressed CYPs for use as reagents for in vitro quantitative phenotyping, and bridging the gap between cDNA-expressed cytochromes and human liver microsomes has been the subject of several recent reviews (Remmel and Burchell, 1993; Crespi, 1995; Crespi and Penman, 1997; Rodrigues, 1999).

The turnover number of CYPs in human liver microsomes is affected by several factors that are biochemically distinct from the CYP enzyme itself. Examples include the accessory electron-transfer proteins NADPH cytochrome P450-oxidoreductase (OR) and cytochromeb5, membrane lipid composition, and ionic strength of the in vitro incubation matrix (Remmel and Burchell, 1993). If the goal of in vitro studies on cDNA-expressed CYPs is to extrapolate the results to human liver microsomal drug metabolism, the experimental conditions with respect to such accessory non-CYP factors should be parallel in the two systems (Remmel and Burchell, 1993). Although factors like ionic strength are easily controlled to ensure similar conditions in both systems, accessory protein concentrations similar to human liver microsomes may not be achievable and thus may well differ between microsomes containing cDNA-expressed CYPs and human liver microsomes. Moreover, heterologously expressed systems contain a single CYP interacting with the accessory proteins, whereas in liver microsomes multiple CYPs may compete for interaction with the accessory proteins. These considerations introduce additional complexity in bridging the gap between expression systems and human liver microsomes, thereby complicating in vitro-in vivo scaling of metabolic rates and relative contributions of the various isoforms.

The substrate concentration-velocity relationship for a hepatic drug biotransformation catalyzed by multiple CYPs can in principle be described as a linear combination of velocity functions for each CYP isoform [vi(s)] weighted for the relative hepatic abundance of the respective isoforms (Ai):V(s)=∑i=1n Aivi(s) Equation 1The relative contribution of each isoform can then be determined as follows:fi(%)=Aivi(s)∑i=1n Aivi(s)×100 Equation 2Enzyme kinetic studies using cDNA-expressed human CYPs are being increasingly used to define the functionsvi(s) for whatever individual CYPs biotransform a drug. Such analyses indicate the relative affinities and capacities of the CYPs contributing to a biotransformation. However, the relative contribution of each enzyme cannot be estimated unless the results incorporate the scaling factors (Ai).

Although immunoquantified levels of CYP isoforms in human liver microsomes (Shimada et al., 1994; Code et al., 1997; Ekins et al., 1998; Lasker et al., 1998; Stresser and Kupfer, 1999) are used as estimates of Ai (Yamazaki et al., 1997;Rodrigues, 1999; Shimada et al., 1999), the capacity (turnover number) of an enzyme determined from kinetic studies on cDNA-expressed CYP isoforms may be different from that of the liver microsomal counterpart (Crespi, 1995; Crespi and Miller, 1997; Crespi and Penman, 1997). Thus,Ai should not only reflect the relative abundance of isoform i in human liver microsomes but also account for turnover number differences between liver microsomes and the heterologous expression system. One such estimate of the scaling factorAi is the relative activity factor (RAF) (Crespi, 1995; Crespi and Penman, 1997).

RAFs are determined for specific CYPs by comparing the rate of an isoform-specific index reaction at saturating substrate concentrations in human liver microsomes (Vmax for liver microsomes) to the rate of the same reaction catalyzed by the specific cDNA-expressed CYP under identical conditions (Vmax for cDNA-expressed enzyme):RAFisoform=Mean Vmax for isoform­specific reaction in human liver microsomesVmax for isoform­specific reaction by cDNA expressed isoform Equation 3When the numerator is expressed in picomoles metabolite per milligram of protein per minute and the denominator as picomoles of metabolite per picomole of CYP per minute, the units of RAF are picomoles of CYP per milligram of protein, which can be compared with the immunoquantified CYP contents. RAFs have been used as estimates ofAi in reaction phenotyping (Kobayashi et al., 1997; Nakajima et al., 1999; Roy et al., 1999; von Moltke et al., 1999; Huang et al., 2000), but their quantitative difference from immunoquantified CYP levels is not fully established. In addition, it is not clear whether RAFs determined for a given isoform are independent of the choice of index substrate.

The objectives of the present study were to investigate the biochemical bases of scaling from heterologously expressed CYPs to human liver microsomes and to compare RAF estimates with immunoquantified CYP content for multiple CYP isoforms using lymphoblast-expressed enzymes. We have also tested the substrate-independence assumption of the RAF approach using multiple index substrates of CYP1A2, CYP3A4, and CYP2D6. In addition, we have compared the OR:CYP and cytochromeb5:CYP ratios in human liver microsomes to those in lymphoblast microsomes containing cDNA-expressed CYPs to provide an explanation for differences between RAFs and immunoquantified protein levels.

Materials and Methods

Human Liver Microsomes and Heterologously Expressed CYP Isoforms.

Liver samples, obtained from the International Institute for the Advancement of Medicine (Exton, PA) or the Liver Tissue Procurement and Distribution Service (University of Minnesota), were from 12 different transplant donors (L1–L12) with no known liver disease. The donor population (median age 27 years, range 3–50 years; 5 females and 7 males) was balanced with respect to age, sex, race, smoking habits, and alcohol consumption. The tissue was partitioned and kept at −80°C until the time of microsome preparation as described previously (von Moltke et al., 1993, 1994).

Microsomes from cDNA-transfected human lymphoblastoid cells expressing CYP 1A2, 2B6, 2C19, 2D6, or 3A4 (Crespi, 1995) were purchased from Gentest Corporation (Woburn, MA), aliquoted and stored at −80°C, and thawed on ice before use. Lymphoblast-expressed CYPs 3A4 and 2D6 used in the study were coexpressed with OR, whereas the activities of lymphoblast-expressed CYPs 1A2, 2B6, and 2C19 were supported by endogenous levels of reductase native to the host cell line. Microsomal protein concentrations and CYP content were provided by the manufacturer.

Antibodies and Quantitative Western Blotting.

Concentrations of CYP1A2, 2B6, 2C19, 2D6, and 3A4/5 in human liver microsomal preparations from livers L1 to L12 were determined by quantitative Western blotting (Perloff et al., 1999). Microsomal protein (varying amounts of lymphoblast-expressed CYP standards and an optimal amount of liver microsomal protein) was denatured for 5 min at 100°C in 100 mM Tris buffer (pH 6.8) containing 10% glycerol, 2% β-mercaptoethanol, 2% SDS, and 5 μg/ml pyronin Y. Protein was separated by SDS-polyacrylamide gel electrophoresis in precast 7.5% polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) in 25 mM Tris/0.192 M glycine/0.1% SDS running buffer (pH 8.3) and transferred to Immobilon-P paper (0.45-μm pore size; Millipore, Bedford, MA) by electroblotting at 100 V for 1 h in 25 mM Tris/0.192 M glycine/20% methanol transfer buffer. Blots were blocked, incubated with primary antibody for 1 h, washed, reblocked (for CYPs 3A4 and 1A2 blots only), incubated with HRP-labeled secondary antibody for 1 h, washed again, and the bound HRP signal was activated by enhanced chemiluminescence using the Super Signal Cl-HRP substrate system (Pierce, Rockford, IL). All postantibody washings were done three times (5 min each) in TBS (0.15 M NaCl, 0.04 M Tris Cl, pH 7.7) containing 0.06% Tween 20 (TBS-Tween), with the exception of the final two washes before activation of the HRP signal, which were done in TBS alone. Blots were exposed to film, developed, and quantified by computer-aided densitometry (IMAGE 1.62 image analysis software, National Institutes of Health). A calibration curve of integrated band intensity (the product of band area and band intensity; Y) versus the quantity of CYP standard in picomoles (X) was generated and fit to an empirically determined equation (Y = mX + c for CYPs 1A2 and 2C19; Y = m ln X + cfor CYPs 2B6 and 3A4; Y = mX + cor Y = AXB for CYP2D6, where m and c are slope and intercept terms, respectively). Integrated band densities of liver microsomal samples were used to determine the concentration of CYP per milligram of microsomal protein relative to the calibration curve. Specific conditions of the Western blot assays (electrophoretic conditions, blocking conditions, and antibody dilutions) for each CYP isoform are given in Table 1. All antibodies recognized the target CYP isoform with minimal cross-reactivity, based on data from their manufacturers for the CYP2D6, 3A, and 1A antibodies and specificity studies performed in our laboratory for CYP2B6 and 2C19 antibodies. Although the CYP2C19 antibody showed a minor cross-reactivity with CYP2A6, this did not affect the analysis because the two proteins were electrophoretically resolved under the conditions used.

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Table 1

Western blotting conditions for human liver microsomal CYPs 1A2, 2B6, 2C19, 3A, and 2D6

In Vitro Metabolic Incubations and Metabolite Analysis.

Table 2 lists the incubation conditions and metabolite analysis methodology for index reactions used for determination of RAF estimates for CYPs 1A2, 2B6, 2C19, 3A, and 2D6.

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Table 2

Summary of incubation conditions and analytical methods for in vitro metabolism assays

Incubations of index substrates with human liver microsomes and lymphoblast microsomes containing cDNA-expressed CYPs were performed using standard procedures as described earlier for other reactions (von Moltke et al., 1993, 1994; Schmider et al., 1996). Reactions were performed at 37°C in 50 mM KH2PO4 (pH 7.4) containing 475 μg/ml MgCl2, 0.5 mg/ml β-NADP+, 1 mg/ml isocitric acid, and 0.32 unit/ml isocitric dehydrogenase (NADPH regenerating system), in a total volume of 250 μl. Microsomal protein concentrations and reaction times were chosen to be in the linear range and to minimize substrate consumption. Reactions were stopped by addition of 100 μl of acetonitrile with the exception of bupropion hydroxylation, which was stopped by the addition of 50 μl of 1 M HCl, and diazepam 3-hydroxylation, which was stopped by the addition of 100 μl of acetonitrile containing 2% (w/v) butylated hydroxytoluene to ensure metabolite stability. Internal standard was added, and the incubations were processed for chromatographic analysis by HPLC (Waters Associates, Milford, MA); diazepam incubations were analyzed by gas chromatography with electron-capture detection (5890 Series II GC System, Hewlett-Packard, Palo Alto, CA) after liquid-liquid extraction into 1.5 ml of toluene containing 1.5% (v/v) isoamyl alcohol.

Enzyme Kinetic Methods and Determination of RAF Estimates.

Standard nonlinear regression-based enzyme kinetic methods were used in the determination of Vmax values for ethoxyresorufin O-deethylation (EROD), methoxyresorufinO-demethylation (MROD), S-mephenytoin 4-hydroxylation, and diazepam 3-hydroxylation, both for human liver microsomes (L1–L12) and for the respective lymphoblast-expressed isoforms. RAF estimates were then calculated using eq. 3. The determination of CYP2C19 RAFs for this panel of 12 livers (Venkatakrishnan et al., 1998c) and the evidence for CYP2C19 poor metabolizing phenotype of livers L11 and L12 (Venkatakrishnan et al., 1998a,c) have been described previously. Although a Michaelis-Menten model best described the kinetics of EROD and MROD, the kinetics of diazepam 3-hydroxylation by human liver microsomes and lymphoblast-expressed CYP3A4 were fitted to a one enzyme Hill model (based on convex Eadie-Hofstee plots indicative of autoactivation by substrate). Phenacetin O-deethylation (POD) (von Moltke et al., 1996a) and nortriptyline E-10 hydroxylation (Venkatakrishnan et al., 1999b) displayed biphasic kinetics in human liver microsomes and were described by a two-enzyme model with a high-affinity Michaelis-Menten component and a linear approximation of the low affinity component. The Vmax of the high-affinity components of POD and nortriptyline E-10 hydroxylation [previously validated as indices of CYP1A2 (Venkatakrishnan et al., 1998b) and CYP2D6 (Venkatakrishnan et al., 1999b) activity, respectively] were used in the calculation of the CYP1A2 and CYP2D6 RAFs, respectively. In addition to the above-described kinetic method, the CYP1A2 Vmax of POD in human liver microsomes was also determined independently by an inhibition method, as the α-naphthoflavone-sensitive component. For this, the difference between human liver microsomal POD rate at 1000 μM phenacetin and the rate in the presence of 2 μM α-naphthoflavone [validated to specifically and completely inhibit CYP1A2 (Venkatakrishnan et al., 1999a)] was used as the Vmax of the CYP1A2 component of POD, for determination of the POD RAF. For determination of CYP2D6 RAFs with bufuralol and desipramine as index substrates, the quinidine (5 μM)-inhibitable components of reaction rate were used, at substrate concentrations of 160 and 100 μM, respectively. For determination of CYP3A RAFs using trazodone (Rotzinger et al., 1998;Zalma et al., 2000) and triazolam (von Moltke et al., 1996b) as index substrates, and for determination of CYP2B6 RAFs using bupropion (Hesse et al., 2000), reaction rates were measured at saturating substrate concentrations (1000, 1000, and 250 μM, respectively).

Measurement of OR Concentrations.

Concentrations of OR in human liver microsomes and in microsomes from lymphoblastoid cells expressing CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 were determined using an activity assay that spectrophotometrically measures the rate of NADPH-mediated reduction of cytochrome c(Phillips and Langdon, 1962; Pearce et al., 1996). The 1-ml sample and reference cuvettes each contained 330 mM KH2PO4 (pH 7.6), 1 mM KCN, 50 μM cytochrome c (Sigma, St. Louis, MO) and microsomal protein (25–100 μg). Reactions were started by the addition of 10 μl of a 4.2 mM solution of β-NADPH (Sigma) to the sample cuvette. The rate of reduction of cytochrome c was determined as the slope of the initial linear part of the curve describing the time-dependent increase in absorbance at 550 nm. Spectra were recorded at room temperature with a dual beam spectrophotometer (Uvikon, Kontron, Zurich). Human liver and lymphoblast microsomal OR concentrations were determined in reference to a calibration curve generated using 0.8, 1.6, 3.2, and 6.4 pmol of purified recombinant human OR (Oxford Biomedical Research, Oxford, MI).

Measurement of Cytochrome b5Concentrations.

The concentration of cytochrome b5 in human liver microsomes and microsomes from lymphoblastoid cells expressing CYPs 1A2, 2B6, 2C19, 2D6, and 3A4 were determined from the difference spectrum between NADH-reduced and oxidized microsomes (Estabrook and Werringloer, 1978; Pearce et al., 1996). Microsomes were diluted to 1 mg/ml in 100 mM KH2PO4, pH 7.4, and divided between two 1-ml cuvettes. After a baseline of equal light absorbance was obtained between 400 and 500 nm, 5 μl of 20 mM β-NADH (Sigma) was added to the sample cuvette, and the difference spectrum was recorded. The concentration of cytochromeb5 was determined from the absorbance difference between 410 nm (trough) and 425 nm (peak), based on an extinction coefficient of 185 mM−1cm−1.

Reconstitution of CYP1A2 with OR.

The effects of increasing molar ratios of OR to CYP1A2 on the rates of EROD and POD were studied by reconstitution experiments (Shimada and Yamazaki, 1998) with purified Escherichia coli-expressed human CYP1A2 (Panvera, Madison, WI) and purified recombinant human OR (Oxford Biomedical Research). CYP1A2 (6.25 pmol) was mixed with varying amounts of OR (to yield molar OR:CYP ratios of 0.05, 0.1, 0.5, 1, 2, 5, and 10) and 5 μg ofl-α-dilauroyl-sn-glycero-3-phosphocholine (added from a 1 mg/ml solution, freshly prepared in 10 mM KH2PO4, pH 7.4, by sonication at 4°C) and incubated at 25°C for 5 min with shaking, in a total volume of 55 μl. Reconstituted CYP1A2-OR-lipid mixtures were added to reaction tubes containing substrate (1000 μM phenacetin or 2500 nM ethoxyresorufin) and the prewarmed NADPH regenerating system and incubated with shaking at 37°C. EROD reactions using reconstituted CYP1A2 were performed for 1 min to ensure linearity of product formation rate with time and to avoid direct one-electron reduction of the resorufins to the nonfluorescent semiquinone imine form by OR (Schmalix et al., 1996). Incubations were processed for analysis as described earlier for microsomal incubations.

Statistical Analyses.

Multiple linear regression and nonparametric Spearman rank order correlation analyses were performed using the SigmaStat software (SPSS Inc., Chicago, IL). Hypotheses regarding population median ratios were tested using one-sample Wilcoxon signed rank tests.

Results

Immunoquantified CYP Contents, Lymphoblast RAF Estimates, and RAF:CYP Content Ratios.

Representative immunoblots and calibration curves are shown in Fig.1. Median values of immunoreactive content and RAF estimates are provided in Table3. The median rather than arithmetic mean was chosen as a measure of central tendency due to the skewed nature of the distributions and wide range of values in the sample of 12 livers, especially for CYPs 2B6 and 3A. The ratio of lymphoblast RAF estimate to the immunoquantified level of each CYP isoform for the sample of 12 livers is presented in Fig.2.

Figure 1
Figure 1

Quantitative Western blot analysis of CYPs 1A2, 2B6, 2C19, and 3A in human liver microsomes.

Representative blots are shown on the left and a representative calibration curve on the right. Only liver microsomal band intensities in the range of the calibration curve were used for quantification. CYP1A2 blot: Lanes 1 through 4 are calibration standards, with 1, 0.5, 0.25, and 0.125 pmol of lymphoblast-expressed CYP1A2 loaded per lane, respectively. Lanes 5 through 10 are varying amounts (6–24 μg) of microsomal protein from livers L1 through L6, respectively. Protein loading was normalized for CYP1A2 activity (570 pmol of ethoxyresorufinO-deethylated per minute at a substrate concentration of 2500 nM). Note the variable band intensity in lanes 5 through 10 despite activity-normalized loading of microsomal protein, indicating interindividual variability in turnover number of human liver microsomal CYP1A2. CYP2B6 blots: For blot 1 (lanes 1–9), lanes 1 though 5 are calibration standards, with 0.1, 0.25, 0.5, 1, and 2.5 pmol of lymphoblast-expressed CYP2B6 loaded per lane, respectively. Lanes 6, 7, 8, and 9 were loaded with 10 μg of microsomal protein from livers L1, L2, L3, and L10, respectively. For blot 2 (lanes 1′–10′), lanes 5′ through 10′ are calibration standards, with 2.5, 1, 0.5, 0.25, 0.1, and 0.05 pmol of lymphoblast-expressed CYP2B6 loaded per lane, respectively. Lanes 1′, 2′, 3′, and 4′ were loaded with 50 μg of microsomal protein from livers L8, L7, L6, and L5, respectively. CYP2C19 blot: Lanes 7 through 11 are calibration standards, with 0.03125, 0.0625, 0.125, 0.25, and 0.5 pmol of lymphoblast-expressed CYP2C19 loaded per lane, respectively. Lanes 1 through 6 were loaded with 15 μg of microsomal protein from livers L12, L11, L4, L3, L2, and L1, respectively. Note the lack of immunodetectable CYP2C19 in livers L12 and L11 (poor metabolizers ofS-mephenytoin). CYP3A blot: Lanes 1 through 4 are calibration standards, with 4, 2, 1, and 0.5 pmol of lymphoblast-expressed CYP3A4 loaded per lane, respectively. Lanes 5 through 10 are varying amounts (2–21 μg) of microsomal protein from livers L1 through L6, respectively. Protein loading was normalized for CYP3A activity (13 pmol of triazolam 1-hydroxylated per minute at a substrate concentration of 1000 μM). Note the variable band intensity in lanes 5 through 10 despite activity-normalized loading of microsomal protein, indicating interindividual variability in turnover number of human liver microsomal CYP3A. CYP2D6 blot: Lanes 7 through 11 are calibration standards, with 0.5, 0.25, 0.125, 0.0625, and 0.03125 pmol of lymphoblast-expressed CYP2D6 loaded per lane, respectively. Lanes 1 through 6 were loaded with 5 to 25 μg of microsomal protein from livers L12, L11, L10, L9, L8, and L7, respectively. Note the lack of immunodetectable CYP2D6 in liver L8 (poor metabolizer phenotype).

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Table 3

Immunoquantified CYP concentrations and RAF estimates in human liver microsomes

Figure 2
Figure 2

Ratio of RAF estimate to immunoquantified CYP content for CYPs 1A2, 2B6, 2C19, and 3A.

Multiple index reactions were used in the determination of CYP1A2 and -3A RAFs. BUP, bupropion; SMH, S-mephenytoin 4-hydroxylation. Hollow circles represent individual human livers, with the heavy dashed line denoting the sample median. The solid line is the line of identity between RAF estimates and immunoquantified protein levels, and the dashed upper and lower control lines denote RAF:CYP content ratios of 3 and 0.33, respectively. Note that the RAF:CYP content ratio for CYPs 1A2 and 2B6 lie above the upper control line, whereas the ratios for CYPs 2C19, 3A, and 2D6 lie between the lower and upper control lines, for the majority of the livers.

With phenacetin as the index substrate, the enzyme kinetic method and inhibition method yielded similar median CYP1A2 RAF estimates of 394 and 380 pmol/mg of protein, respectively. These values were 3- to 4-fold higher than those determined using EROD (124 pmol/mg of protein), and MROD (103 pmol/mg of protein). In addition, all three CYP1A2 RAFs were consistently higher than the immunoquantified CYP1A2 content (median value 19 pmol/mg of protein) in all 12 liver samples (Table 3 and Fig. 2).

CYP2B6 RAF values determined using bupropion as index substrate (161 pmol/mg of protein) were consistently higher than the immunoquantified levels of CYP2B6 (45 pmol/mg of protein) in all 12 liver samples (Table2 and Fig. 2).

CYP2C19 content and RAF values were similar (median values of 20 and 29 pmol/mg of protein, respectively). However, the ratio of RAF to CYP2C19 content was greater than 1 in 9 of 10 livers (with the exception of liver L9, which had a ratio of 0.87). The median RAF:CYP2C19 content ratio (1.48, n = 10) was significantly different from unity.

Median values of CYP3A RAFs determined using triazolam, trazodone, and diazepam were 254, 102, and 125 pmol/mg of protein, respectively (n = 12). Median immunoquantified CYP3A content was 262 pmol/mg of protein. For all three substrates examined, median values of the RAF:3A content ratio (1.35, 0.6, and 0.56 for triazolam, trazodone, and diazepam, respectively) were not significantly different from unity.

Median values of CYP2D6 RAFs determined using bufuralol, desipramine, and nortriptyline were 8.0, 8.9, and 11.6 pmol/mg of protein, respectively (n = 11). Median immunoquantified CYP2D6 content was 10.4 pmol/mg of protein. For all three substrates examined, median values of the RAF:2D6 content ratio (0.68, 0.83, and 0.88 for bufuralol, desipramine, and nortriptyline, respectively) were not significantly different from unity.

Concentrations of Accessory Proteins in Human Liver Microsomes and Lymphoblast Microsomes Containing Heterologously Expressed Cytochromes.

The concentration of OR in human liver microsomes was 69.8 ± 20 pmol/mg of protein (mean ± S.D., n = 12 livers; range 47–103 pmol/mg of protein). In microsomes from human lymphoblastoid cells coexpressing CYP3A4 and OR, the OR concentration was 35 pmol/mg of protein. In microsomes from human lymphoblastoid cells coexpressing CYP2D6 and OR, the OR concentration was 62.5 pmol/mg of protein. In contrast, microsomes from lymphoblastoid cells expressing CYPs 1A2, 2B6, and 2C19 had OR concentrations that were an order of magnitude lower (3.4, 3.7, and 5 pmol/mg of protein, respectively). The molar ratios of OR to CYP in lymphoblast-expressed CYP1A2, -2B6, and -2C19 were 92-, 29-, and 24-fold lower than their respective values (medians) in human liver microsomes, whereas the OR:CYP3A ratio was similar in the two systems (Table4 and top panels of Fig.3). The OR:CYP2D6 ratio in lymphoblast microsomes containing cDNA-expressed CYP2D6 was 3.7-fold lower than the median value in human liver microsomes; however, these values are within the same order of magnitude.

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Table 4

Molar ratios of concentrations of accessory proteins (OR or cytochromeb5) and concentrations of CYPs 1A2, 2B6, 2C19, 3A4, or 2D6 in human liver microsomes, and commercially available lymphoblast microsomes containing cDNA-expressed cytochromes

Figure 3
Figure 3

Accessory protein:CYP ratios in human liver microsomes and lymphoblast microsomes containing cDNA-expressed CYP isoforms.

Top panels show OR:CYP ratios; lower panels show cytochromeb5:CYP ratios. HLM, human liver microsomes; LYM, lymphoblast microsomes. Hollow circles represent individual human livers, with the dashed line denoting the sample median ratio. Solid dots represent ratios in lymphoblast microsomes.

Figure 4 is a representative difference spectrum recorded after NADH reduction of microsomal cytochromeb5, showing an approximately 5-fold difference in trough-to-peak absorbance difference (indicative of cytochrome b5 concentration) between human liver microsomes and lymphoblast microsomes. The concentration of cytochrome b5 in human liver microsomes was 374 ± 128 pmol/mg of protein (mean ± S.D.,n = 12 livers; range 157–569 pmol/mg of protein). Microsomes from lymphoblastoid cells expressing CYPs 1A2, 2B6, 2C19, 3A4 + OR, and 2D6 + OR all had similar cytochromeb5 concentrations (65–76 pmol/mg of protein). The cytochrome b5:CYP ratio in lymphoblast-expressed CYP1A2, 2B6, 2C19, and 2D6 were 29-, 13-, 9-, and 64-fold lower than their respective values (medians) in human liver microsomes, whereas the cytochrome b5:CYP3A ratio was similar in the two systems (Table 4 and bottom panels of Fig.3).

Figure 4
Figure 4

Representative cytochrome b5difference spectra showing a representative human liver (with cytochrome b5 content close to the sample median) and lymphoblast microsomes containing CYP3A4.

Equal microsomal protein concentrations (1 mg/ml) were used for both preparations. Note the approximately 5-fold lower absorbance difference trough (410 nm) to peak (425 nm) (indicative of cytochromeb5 content) in lymphoblast microsomes compared with human liver microsomes.

Association of Human Liver Microsomal Accessory Protein:CYP Content Ratios with RAF:CYP Content Ratios: Correlation Studies.

To test the hypothesis that differences in accessory protein:CYP content ratios between liver microsomes and lymphoblast microsomes may account for the departure of RAFs from immunochemically determined abundance estimates, a statistical approach was used, involving the examination of correlations between OR:CYP ratios or cytochromeb5:CYP ratios (X), and the ratio of RAF to immunoquantified levels of a particular CYP isoform (Y) in the panel of human liver microsomes studied. Due to the skewed nature of the distributions and the relatively small sample size, a nonparametric Spearman rank order correlation analysis was used.

Table 3 provides the Spearman correlation coefficients examining associations between OR:CYP ratios (r1) or cytochrome b5:CYP ratios (r2) and RAF:CYP content ratios for CYPs 1A2, 2B6, 2C19, 3A, and 2D6. Representative scatter plots are shown in Figs. 5(OR) and 6 (cytochrome b5). Significant positive associations were noted between the ratio of OR or cytochromeb5 to CYP isoform content and the ratio of RAF to immunochemically determined CYP concentrations, for CYPs 1A2, 2B6, 3A, and 2D6, but not for CYP2C19.

Figure 5
Figure 5

Representative scatter plots showing the association between OR:CYP ratios and RAF:immunoquantified CYP content ratios in human liver microsomes, for CYPs 1A2 (A), 2B6 (B), 2C19 (C), and 3A (D).

Spearman correlation coefficients (rs values) are provided.

Figure 6
Figure 6

Representative scatter plots showing the association between cytochrome b5:CYP ratios and RAF:immunoquantified CYP content ratios in human liver microsomes, for CYPs 1A2 (A), 2B6 (B), 2C19 (C), and 3A (D).

Spearman correlation coefficients (rsvalues) are provided.

Reconstitution of CYP1A2 Activity: Effect of OR:CYP Molar Ratio.

Rates of both CYP1A2-catalyzed EROD (Fig. 7A) and POD (Fig. 7B) increased with increasing concentrations of OR, at a fixed concentration of CYP1A2. At OR:CYP1A2 ratios of 0.05 (similar to those in lymphoblast microsomes containing CYP1A2), rates of EROD and POD were near the limit of detection. Reaction rates could be reliably measured only at OR:CYP ratios of 0.1 and higher. With both substrates, the metabolic rate (indicative of CYP1A2 turnover number) at OR:CYP ratios of 1 to 10 (parallel to those in human liver microsomes) were 10- to 20-fold higher than the rate at OR:CYP ratios of 0.1 (close to the ratio in lymphoblast microsomes containing CYP1A2).

Figure 7
Figure 7

Reconstitution of CYP1A2 with varying amounts of OR.

A and B, the concentration-dependent effect of increasing molar ratios of OR and CYP1A2 on EROD and POD activity, respectively. C, the effect of the OR:CYP ratio on the ratio of relative POD activity to relative EROD activity. Arrows, typical OR:CYP1A2 ratios in lymphoblast microsomes containing CYP1A2 and in human liver microsomes (0.04 and 3.5, respectively). Note a modest increase in the ratio of relative activities with increasing OR:CYP1A2 ratio, indicating a small degree of substrate dependence in OR requirement.

To test the hypothesis that differences in reductase requirements of EROD and POD may explain in part the substrate dependence of RAF estimates, multiple linear regression analyses were performed. With POD RAF as the dependent variable (Y) and EROD or MROD RAFs (X1) and OR concentrations (X2) as dependent variables, approximately 40% of the variability in POD RAFs was explained by the variability in OR content. Consistent with this observation, the ratio of relative POD activity to relative EROD activity showed a modest increase with increasing OR:CYP1A2 ratio in reconstitution experiments (Fig. 7C), indicating a small difference in reductase requirement for the two substrates.

Discussion

Scaling from heterologously expressed CYPs to human liver microsomes requires the use of appropriate scaling factors (Ai values in eqs. 1 and 2). An accurate scaling factor should reflect both the relative abundance of a particular CYP isoform in human liver as well as the differences in turnover number between the cDNA-expressed CYP and its liver microsomal counterpart. This is the conceptual basis of the RAF scaling approach. Thus, the RAF estimate for a CYP will equal its immunoquantified content in liver microsomes only if the turnover number of the enzyme in heterologously expressed form equals that of its liver microsomal counterpart. The turnover numbers of CYP enzymes is known to depend on the concentrations of accessory electron-transfer proteins, which may well differ between cDNA-expressed systems and human liver. However, there has not yet been a systematic quantitative investigation of differences in CYP turnover number between the two systems. Immunoquantified CYP levels continue to be used as scaling factors (Rodrigues, 1999; Shimada et al., 1999), but they may not represent accurate estimates of AI (Störmer et al., 2000) because they do not incorporate turnover number differences.

The present study has demonstrated that accessory protein:CYP content ratios in some cDNA-expressed CYP preparations are in fact different from those in human liver microsomes and that lymphoblast RAF estimates are significantly different from immunoquantified CYP concentrations for some CYP isoforms. These findings have important implications in bridging the gap between cDNA-expressed CYPs and human liver microsomes and, ultimately, in the accurate determination of relative contributions of individual CYP isoforms to overall clearance of a drug.

Lymphoblast cell lines expressing CYPs 2A6, 2C9, 2D6, 2E1, and 3A4 are engineered to coexpress OR. However, CYPs 1A2, 2B6, and 2C19 have not been successfully coexpressed with OR and are thus dependent on reductase endogenous to the cell line for metabolic activity. Considerable effort was devoted to isolating lymphoblasts coexpressing 1A2, 2B6, and 2C19 with OR; however, stable cell lines with activities higher than the cell lines without OR were not obtained. For the enzymes that could be expressed with OR, differences in CYP expression efficiency also influence the final OR:CYP ratio.

CYP1A2 and 2B6 could not be coexpressed with OR in the lymphoblast system, and the OR:CYP ratios in lymphoblast microsomes containing these cytochromes were over an order of magnitude lower than their respective values in human liver microsomes. Consistent with these observations, the RAF values for CYPs 1A2 and 2B6 were significantly higher than their immunoquantified protein levels. For CYP1A2, it was further established using reconstitution experiments that the OR:CYP ratio differences between liver and lymphoblast microsomes account for the deviation of RAF estimates from immunoquantified CYP1A2 levels. Although cytochrome b5:CYP1A2 ratios in lymphoblast microsomes were also significantly lower than those in human liver microsomes, this should not influence the turnover number of the enzyme because it is established that cytochromeb5 does not stimulate CYP1A2 activity (Yamazaki et al., 1997; Shimada and Yamazaki, 1998).

For CYP2C19, accessory protein:CYP ratios were significantly lower in lymphoblast microsomes than in human liver microsomes, but this was reflected as only a 1.5-fold higher RAF in comparison to immunoquantified protein levels. This suggests that the interaction of CYP2C19 with the flavoproteins is saturated at lower accessory protein:CYP ratios.

OR:CYP2D6 ratios in lymphoblast microsomes and human liver microsomes were within the same order of magnitude. This was consistent with similar values of CYP2D6 RAFs and immunochemical content. As with CYP1A2, cytochrome b5:CYP2D6 ratios in lymphoblast microsomes were significantly lower than those in human liver microsomes. However, this did not affect the CYP2D6 turnover number, a finding consistent with the lack of stimulation of CYP2D6 activity by cytochrome b5 in reconstituted systems (Yamazaki et al., 1997).

Median values of CYP3A RAFs were similar to immunoquantified CYP3A concentrations. This is consistent with the observation that the median values of both OR:CYP3A and cytochromeb5:CYP3A ratios in lymphoblast microsomes were similar to those in human liver microsomes.

A large interindividual variability was noted in the RAF:CYP content ratio in the panel of 12 livers for CYPs 1A2, 2B6, and 3A. This indicates that the turnover number for a specific CYP isoform can be different between individuals. This is clearly evident upon examination of the CYP1A2 and -3A immunoblots in Fig. 1. Protein loading for the liver samples was activity-normalized, yet the immunoreactive band intensities show a large variation. Livers with lower turnover number had lower accessory protein:CYP ratios. These interindividual differences can confound the interpretation of CYP content-based results unless pools of human liver microsomes are used (and such differences are thus averaged) but are taken into account by the nature of the RAF approach. This finding suggests that the interindividual variability in hepatic drug metabolism is attributable to differences in the levels of expression of individual CYPs as well as differences in the levels of accessory electron-transfer proteins in relation to CYP content. The in vivo pharmacokinetic implications of this finding require further investigation.

Although the RAFs are expected, in principle, to be quantitatively different from immunoquantified CYP isoform content (Crespi, 1995;Crespi and Penman, 1997) due to potential differences in turnover number of heterologously expressed CYP isoforms from those of their human liver microsomal counterparts, this has not been demonstrated previously. The present study has demonstrated using lymphoblast-expressed CYP isoforms that some RAFs were identical to immunochemically determined CYP content, whereas others were not.

Unlike immunoquantified levels that only indicate hepatic abundance, RAFs also incorporate differences in turnover number between the cDNA-expressed and liver microsomal CYPs. RAFs, rather than immunoquantified CYP content, are thus better initial estimates of scaling factors (Ai values in eqs. 1 and 2) for bridging the gap between cDNA-expressed CYPs and human liver microsomes (Störmer et al., 2000). Although RAF estimation is somewhat dependent on the choice of index substrate (possibly due to substrate-dependent differences in accessory protein requirement), this factor introduces only a 2- to 4-fold difference in the estimated RAF. The 2- to 4-fold difference is similar to differences in mean specific CYP contents that have been reported in the literature. In contrast, much greater differences between RAFs and immunoquantified protein levels (up to an order of magnitude or higher) are demonstrated here using lymphoblast-expressed CYPs as the model system. Use of immunoquantified CYP levels (in place of RAFs) in conjunction with the in vitro kinetic parameters of cDNA-expressed CYPs may yield inaccurate predictions of relative contributions of the various CYPs to overall human liver microsomal metabolic rate, and thus drug clearance in vivo. In the present study, we identified an underestimation of the contribution of CYP isoforms such as CYP1A2 and 2B6 whose turnover numbers in the lymphoblast expression system are significantly lower than those of their human liver microsomal counterparts. Although the present study was restricted to CYPs expressed in the lymphoblast system, the effects observed should be applicable regardless of the expression system used. Indeed, no system may be capable of achieving physiological levels of accessory proteins across the spectrum of xenobiotic metabolizing CYPs. Therefore, consideration of relative enzymatic activities at some level may be unavoidable.

Footnotes

  • Send reprint requests to: David J. Greenblatt, Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Harrison Avenue, Boston, MA 02111. E-mail: Dj.Greenblatt{at}tufts.edu

  • This work was supported by Grants MH-34223, DA-05258, DA-13209, MH-19924, MH-58435, GM-61834, and RR-00054 from the Department of Health and Human Services. Dr. von Moltke was the recipient of a Scientist Development Award (K21-MH-01237) from the National Institutes of Mental Health. Dr. Court was supported by a Special Emphasis Research Career Award (SERCA) from the National Center for Research Resources, National Institutes of Health (K01-RR-00104).

  • Abbreviations used are::
    CYP
    cytochrome P450
    RAF
    relative activity factor
    OR
    NADPH:cytochrome P450 oxidoreductase
    EROD
    ethoxyresorufin O-deethylation
    MROD
    methoxyresorufin O-demethylation
    POD
    phenacetinO-deethylation
    HRP
    horseradish peroxidase
    TBS
    Tris-buffered saline
    Vmax
    maximal reaction rate at saturating substrate concentrations
    • Received June 19, 2000.
    • Accepted August 22, 2000.

References

  1. ↵
    1. Code EL,
    2. Crespi CL,
    3. Penman BW,
    4. Gonzalez FJ,
    5. Chang TKH,
    6. Waxman DJ
    (1997) Human cytochrome P4502B6: Interindividual hepatic expression, substrate specificity, and role in procarcinogen activation. Drug Metab Dispos 25:985–993.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Crespi CL
    (1995) Xenobiotic-metabolizing human cells as tools for pharmacological and toxicological research. Adv Drug Res 26:179–235.
    OpenUrlCrossRef
  3. ↵
    1. Crespi CL,
    2. Miller VP
    (1997) The R144C change in the CYP2C9*2 allele alters interaction of the cytochrome P450 with NADPH:cytochrome P450 oxidoreductase. Pharmacogenetics 7:203–210.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Crespi CL,
    2. Penman BW
    (1997) Use of cDNA-expressed human cytochrome P450 enzymes to study potential drug-drug interactions. Adv Pharmacol 43:171–188.
  5. ↵
    1. Ekins S,
    2. Vandenbranden M,
    3. Ring BJ,
    4. Gillespie JS,
    5. Yang TJ,
    6. Gelboin HV,
    7. Wrighton SA
    (1998) Further characterization of the expression in liver and catalytic activity of CYP2B6. J Pharmacol Exp Ther 286:1253–1259.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Estabrook RW,
    2. Werringloer J
    (1978) The measurement of difference spectra: Application to the cytochromes of microsomes. Methods Enzymol 52:212–220.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Hesse LM,
    2. Venkatakrishnan K,
    3. Court MH,
    4. von Moltke LL,
    5. Duan SX,
    6. Shader RI,
    7. Greenblatt DJ
    (2000) CYP2B6 mediates the in vitro biotransformation of bupropion to hydroxybupropion: Potential drug interactions with other antidepressants. Drug Metab Dispos 28:1176–1183.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Huang Z,
    2. Roy P,
    3. Waxman DJ
    (2000) Role of human liver microsomal CYP3A4 and CYP2B6 in catalyzing N-dechloroethylation of cyclophosphamide and ifosfamide. Biochem Pharmacol 59:961–972.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Kobayashi K,
    2. Chiba K,
    3. Yagi T,
    4. Shimada N,
    5. Taniguchi T,
    6. Horie T,
    7. Tani M,
    8. Yamamoto T,
    9. Ishizaki T,
    10. Kuroiwa Y
    (1997) Identification of cytochrome P450 isoforms involved in citalopram N-demethylation by human liver microsomes. J Pharmacol Exp Ther 280:927–933.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Lasker JM,
    2. Wester MR,
    3. Aramsombatdee E,
    4. Raucy JL
    (1998) Characterization of CYP2C19 and CYP2C9 from human liver: Respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:16–28.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Nakajima M,
    2. Nakamura S,
    3. Tokudome S,
    4. Shimada N,
    5. Yamazaki H,
    6. Yokoi T
    (1999) Azelastine N-demethylation by cytochrome P-450 (CYP)3A4, CYP2D6, and CYP1A2 in human liver microsomes: Evaluation of approach to predict the contribution of multiple CYPs. Drug Metab Dispos 27:1381–1391.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Pearce RE,
    2. McIntyre CJ,
    3. Madan A,
    4. Sanzgiri U,
    5. Draper AJ,
    6. Bullock PL,
    7. Cook DC,
    8. Burton LA,
    9. Latham J,
    10. Nevins C,
    11. Parkinson A
    (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Biochem Biophys 331:145–169.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Perloff MD,
    2. von Moltke LL,
    3. Cotreau MM,
    4. Greenblatt DJ
    (1999) Unchanged CYP 3A expression and metabolism of midazolam, triazolam, and dexamethasone in mdr(−/−) mouse liver microsomes. Biochem Pharmacol 57:1227–1232.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Phillips AH,
    2. Langdon RG
    (1962) Hepatic triphosphopyridine nucleotide cytochrome c reductase: Isolation, characterization and kinetic studies. J Biol Chem 237:2652–2660.
    OpenUrlFREE Full Text
  15. ↵
    1. Remmel RP,
    2. Burchell B
    (1993) Validation and use of cloned, expressed human drug-metabolizing enzymes in heterologous cells for analysis of drug metabolism and drug-drug interactions. Biochem Pharmacol 46:559–566.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Rodrigues AD
    (1999) Integrated cytochrome P450 reaction phenotyping: Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem Pharmacol 57:465–480.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Rotzinger S,
    2. Fang J,
    3. Baker GB
    (1998) Trazodone is metabolized to m-chlorophenylpiperazine by CYP3A4 from human sources. Drug Metab Dispos 26:572–575.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Roy P,
    2. Yu LJ,
    3. Crespi CL,
    4. Waxman DJ
    (1999) Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 27:655–666.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Schmalix WA,
    2. Lang D,
    3. Schneider A,
    4. Bocker R,
    5. Greim H,
    6. Doehmer J
    (1996) Stable expression and coexpression of human cytochrome P450 oxidoreductase and cytochrome P450 1A2 in V79 Chinese hamster cells: Sensitivity to quinones and biotransformation of 7-alkoxyresorufins and triazines. Drug Metab Dispos 24:1314–1319.
    OpenUrlAbstract
  20. ↵
    1. Schmider J,
    2. Greenblatt DJ,
    3. von Moltke LL,
    4. Harmatz JS,
    5. Duan SX,
    6. Karsov D,
    7. Shader RI
    (1996) Characterization of six in vitro reactions mediated by human cytochrome P450: Application to the testing of cytochrome P450-directed antibodies. Pharmacology 52:125–134.
    OpenUrlPubMed
  21. ↵
    1. Shimada T,
    2. Tsumura F,
    3. Yamazaki H
    (1999) Prediction of human liver microsomal oxidations of 7-ethoxycoumarin and chlorzoxazone with kinetic parameters of recombinant cytochrome P-450 enzymes. Drug Metab Dispos 27:1274–1280.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Phillips IR
    1. Shimada T,
    2. Yamazaki H
    (1998) Cytochrome P450 reconstitution systems. in Cytochrome P450 Protocols, ed Phillips IR (Humana Press, Totowa, NJ), pp 85–93.
  23. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Störmer E,
    2. von Moltke LL,
    3. Greenblatt DJ
    (2000) Scaling drug biotransformation data from cDNA-expressed CYP to human liver: A comparison of relative activity factors and human liver abundance in studies of mirtazapine metabolism. J Pharmacol Exp Ther 295:793–801.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Stresser DM,
    2. Kupfer D
    (1999) Monospecific antipeptide antibody to cytochrome P-450 2B6. Drug Metab Dispos 27:517–525.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Venkatakrishnan K,
    2. Greenblatt DJ,
    3. von Moltke LL,
    4. Schmider J,
    5. Harmatz JS,
    6. Shader RI
    (1998a) Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: Dominance of CYP 2C19 and 3A4. J Clin Pharmacol 38:112–121.
    OpenUrlPubMed
  27. ↵
    1. Venkatakrishnan K,
    2. von Moltke LL,
    3. Greenblatt DJ
    (1998b) Human cytochromes P450 mediating phenacetin O-deethylation in vitro: Validation of the high-affinity component as an index of CYP1A2 activity. J Pharm Sci 87:1502–1507.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Venkatakrishnan K,
    2. von Moltke LL,
    3. Greenblatt DJ
    (1998c) Relative quantities of catalytically active CYP 2C9 and 2C19 in human liver microsomes: Application of the relative activity factor approach. J Pharm Sci 87:845–853.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Venkatakrishnan K,
    2. von Moltke LL,
    3. Greenblatt DJ
    (1999a) CYP2C9 is a principal low-affinity phenacetin O-deethylase: Fluvoxamine is not a specific CYP1A2 inhibitor (Letter). Drug Metab Dispos 27:1519–1520.
    OpenUrlFREE Full Text
  30. ↵
    1. Venkatakrishnan K,
    2. von Moltke LL,
    3. Greenblatt DJ
    (1999b) Nortriptyline E-10-hydroxylation in vitro is mediated by human CYP2D6 (high affinity) and CYP3A4 (low-affinity): Implications for interactions with enzyme-inducing drugs. J Clin Pharmacol 39:567–577.
    OpenUrlCrossRefPubMed
  31. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Cotreau-Bibbo MM,
    4. Duan SX,
    5. Harmatz JS,
    6. Shader RI
    (1994) Inhibition of desipramine hydroxylation in vitro by serotonin-reuptake-inhibitor antidepressants, and by quinidine and ketoconazole: A model system to predict drug interactions in vivo. J Pharmacol Exp Ther 268:1278–1283.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Duan SX,
    4. Schmider J,
    5. Kudchadker L,
    6. Fogelman SM,
    7. Harmatz JS,
    8. Shader RI
    (1996a) Phenacetin O-deethylation by human liver microsomes in vitro: Inhibition by chemical probes, SSRI antidepressants, nefazodone, and venlafaxine. Psychopharmacology 128:398–407.
    OpenUrlCrossRefPubMed
  33. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Grassi JM,
    4. Granda BW,
    5. Venkatakrishnan K,
    6. Duan SX,
    7. Fogelman SM,
    8. Harmatz JS,
    9. Shader RI
    (1999) Citalopram and desmethylcitalopram in vitro: Human cytochromes mediating transformation, and cytochrome inhibitory effects. Biol Psychiatry 46:839–849.
    OpenUrlCrossRefPubMed
  34. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Harmatz JS,
    4. Duan SX,
    5. Harrel LM,
    6. Cotreau-Bibbo MM,
    7. Pritchard GA,
    8. Wright CE,
    9. Shader RI
    (1996b) Triazolam biotransformation by human liver microsomes in vitro: Effects of metabolic inhibitors, and clinical confirmation of a predicted interaction with ketoconazole. J Pharmacol Exp Ther 276:370–379.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. von Moltke LL,
    2. Greenblatt DJ,
    3. Harmatz JS,
    4. Shader RI
    (1993) Alprazolam metabolism in vitro: Studies of human, monkey, mouse, and rat liver microsomes. Pharmacology 47:268–276.
    OpenUrlPubMed
  36. ↵
    1. Yamazaki H,
    2. Inoue K,
    3. Shaw PM,
    4. Checovich WJ,
    5. Guengerich FP,
    6. Shimada T
    (1997) Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: Effects of contents of these two forms in individual human samples. J Pharmacol Exp Ther 283:434–442.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Zalma A,
    2. von Moltke LL,
    3. Granda BW,
    4. Harmatz JS,
    5. Shader RI,
    6. Greenblatt DJ
    (2000) In vitro metabolism of trazodone by CYP3A: Inhibition by ketoconazole and human immunodeficiency viral protease inhibitors. Biol Psychiatry 47:655–661.
    OpenUrlCrossRefPubMed
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Comparison between Cytochrome P450 (CYP) Content and Relative Activity Approaches to Scaling from cDNA-Expressed CYPs to Human Liver Microsomes: Ratios of Accessory Proteins as Sources of Discrepancies between the Approaches

Karthik Venkatakrishnan, Lisa L. von Moltke, Michael H. Court, Jerold S. Harmatz, Charles L. Crespi and David J. Greenblatt
Drug Metabolism and Disposition December 1, 2000, 28 (12) 1493-1504;
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