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Vol. 29, Issue 3, 319-325, March 2001
-Acetylmethadol (LAAM)
N-Demethylation and Dextromethorphan
O-Demethylation
Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
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Abstract |
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 Top
 Abstract
 Introduction
Results and Discussion
References
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We examined three primary variables in the preparation of human
liver microsomes. In three experiments, each using three livers, we
manipulated 1) the force of the first centrifugation (9,000, 10,500, or
12,000g); 2) the presence of sucrose in the
homogenization buffer; and 3) the number of homogenizing strokes (6, 8, or 10). Sedimentation plots for the marker enzymes succinate
dehydrogenase, NADPH cytochrome P450 reductase (reductase), and
glutathione S-transferase in the resulting
premicrosomal, microsomal, and cytosolic fractions suggest that
enhanced purity of microsomes can be obtained by reducing force of
centrifugation, including sucrose, and increasing the number of
homogenization strokes. Each microsomal fraction was also assayed for
protein content, cytochrome P450, NADH cytochrome b5 reductase, cytochrome
b5, absorbance at 420, p-nitrophenol hydroxylation, tolbutamide hydroxylation,
dextromethorphan N- and O-demethylation,
glucuronidation of morphine and 1-naphthol, and ester cleavage of
p-nitrophenolacetate. These microsomal indicators were ranked and tested for statistical differences. The use of 9000g statistically increased optimal recovery (per gram
of liver) and specific activity (per milligram of protein). The
inclusion of sucrose improved activity specific to reductase activity.
Ten homogenization strokes improved activity specific to reductase activity. Substrate-dependent activities of dextromethorphan
O-demethylation to dextrorphan and the
N-demethylation of l-
-acetylmethadol
(LAAM) to norLAAM and dinorLAAM were compared in microsomes prepared with or without sucrose and microsomes prepared using 9,000 or 12,000g force, respectively. No significant differences
were found in the concentration-dependent activities. Variation of the
methods used to prepare human liver microsomes can significantly affect the recovery and specific activity of microsomal components; however, they do not appear to affect enzyme kinetics.
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Introduction |
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Top
Abstract
Introduction
Results and Discussion
References
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Human
liver microsomes (HLM2) are used widely to
characterize the role of cytochrome P450s (P450) and other enzymes in
drug metabolism. The generalized, differential centrifugation procedure used to prepare HLM is as follows. Typically, liver samples are homogenized and centrifuged at a lower force to form a crude pellet of
cell debris, nuclei, peroxisomes, lysosomes, and mitochondria (premicrosomal pellet). The resulting supernatant is then centrifuged at a higher force to precipitate the microsomes. The microsomal pellet
is resuspended in a final suspension buffer and is then ready for use
in incubation studies. There are, however, many different procedural
variables associated with this preparation (Table
1) (Boobis et al., 1980
; Raucy and
Lasker, 1991; Kharasch and Thummel, 1993; Guengerich, 1994; Rodrigues
et al., 1994). These variations include the number of strokes, or
passes, used to homogenize the liver samples (e.g., 4-8); the forces
with which the samples are centrifuged (e.g., the centrifuge force for
the first spin ranges from 9,000g for 20 min to
18,000g for 10 min and between 100,000 and
143,000g for 60-90 min for the second spin); and content of
the homogenization and final suspension buffers (EDTA, potassium
chloride, glycerol). While not presented in Table 1, inclusion of
sucrose in the homogenization buffer was considered critical in initial
experiments on differential centrifugation (de Duve, 1971), and it is
used in the preparation of microsomes in experimental animals (Papac
and Franklin, 1988). In some procedures, steps are repeated for more
thorough extraction. Relative volumes, concentrations, and dilutions of
the samples also vary.
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In this study, we examined three variables in the preparation of HLM
that we felt might have the greatest impact on microsomal recovery and
purity. We manipulated the following HLM preparatory variables: the
force of the first centrifugation, the presence of sucrose in the
homogenization buffer, and the number of homogenizing strokes. Cell
fractions were evaluated for separation of marker enzymes. The
microsomal data were analyzed for 12 positive indicators of microsomal
purity (protein content, NADPH cytochrome P450 reductase, P450, NADH
cytochrome b5 reductase, cytochrome
b5, p-nitrophenol hydroxylation,
tolbutamide hydroxylation, dextromethorphan N- and
O-demethylation, glucuronidation of morphine and 1-naphthol, and ester cleavage of p-nitrophenolacetate) and three
negative indicators of microsomal purity (absorbance at 420, succinate dehydrogenase, and glutathione S-transferase). The ideal
fractionation protocol would maximize specific activity and recovery of
endoplasmic reticulum proteins, minimize mitochondrial and cytosolic
enzymes, and minimize denaturation. In two preparations exhibiting
significant differences, the kinetics of two P450-mediated reactions,
the P450 2D6-specific O-demethylation of dextromethorphan
(Kupfer et al., 1984; Schmider et al., 1997) and the P450 3A4
N-demethylation of l--acetylmethadol (LAAM)
(Moody et al., 1997), were measured to determine whether purity
influences kinetic variables in crude microsomal fractions. It is
important to understand how current techniques used to prepare HLM may
compare and how to improve microsomal purity.
Experimental Procedures
Materials.
p-Nitrophenol was obtained from Aldrich (Milwaukee, WI).
Hydroxytolbutamide was provided by Hoechst AG (Frankfurt, Germany). Dextrorphan-d-tartrate was obtained from ICN (Irvine, CA).
1-Naphthol was obtained from J.T. Baker, Inc. (Phillipsburg, NJ).
Morphine was obtained from Merck and Co., Inc. (West Point, PA).
Chlorpropamide was obtained from Pfizer (Brooklyn, NY). Levallorphan
tartrate and 3-methoxymorphinan were obtained from RBI (Natick, MA).
Bovine serum albumin, 1-chloro-2,4-dinitrobenzene, cytochrome
C, 2,6-dichloroindolephenol, dextromethorphan, Folin and
Ciocalteu's phenol reagent, D-glucose 6-phosphate, glucose-6-phosphate dehydrogenase, glutathione, NADP, 
-NADPH, -NADH, p-nitrophenolacetate, potassium
ferricyanide, sodium dithionite, succinic acid, sucrose, tolbutamide,
trichloroacetic acid, and UDP-glucuronic acid were obtained from Sigma
(St. Louis, MO). The liver samples were obtained from the International
Institute for the Advancement of Medicine (Exton, PA) (Table
2).
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HLM Preparation.
To allow for sufficient microsomes to perform all assays, three livers
were prepared for each of the three experiments (the force of the first
centrifugation, homogenization buffer, and homogenizing strokes).
Approximately 10 g of liver per experimental treatment was allowed
to thaw in a room temperature homogenization buffer (0.1 M potassium
phosphate buffer, pH 7.4, containing 0.125 M potassium chloride and 1.0 mM EDTA). After transfer to 25 ml of chilled homogenization buffer
(plus or minus 0.25 M sucrose in the buffer experiment), livers were
minced thoroughly with scissors and homogenized with 10 strokes (6, 8, or 10 strokes in the strokes experiment) using a Teflon-glass
homogenizer (870 rpm). Strokes were even and steady, lasting
approximately 15 s for passage, except for the first two strokes
where greater pressure and time were spent on material on the bottom of
the glass tube. The tube was submersed in a small bucket of ice and
water during all homogenization. The homogenate was diluted to 4 volumes of sample weight (approximately 40 ml). The samples then were
centrifuged at 12,000g (9,000, 10,500, or 12,000g
in the force experiment) in a Sorvall RC-5B with a Sorvall
SA-600 rotor for 20 min (Sorvall, Newton, CT). The supernatant from the
first centrifugation was removed, the mitochondrial pellet was
resuspended in 25 ml, and the centrifugation was repeated. The
supernatants were combined and centrifuged at 138,000g in a
Sorvall Ultra Pro 80 with a Sorvall T-1270 rotor for 60 min. The upper
lipid layer was removed and the cytosolic supernatant collected. The
microsomal pellet was resuspended in 0.125 M KCl, 0.1 M Tris (pH 7.4)
with three homogenization strokes, and the 138,000g
centrifugation for 60 min was repeated. The microsomal pellet was
resuspended in incubation buffer with six strokes and brought to a
final volume of 26 ml. Samples were stored at 
70°C.
Assays.
All resulting liver fractions (premicrosomal, microsomal, and cytosolic
fractions) were subjected to the following four spectrophotometric assays performed in duplicate: protein content (Lowry et al., 1951),
NADPH cytochrome P450 reductase (Masters et al., 1967), succinate
dehydrogenase (Bachmann et al., 1966), and glutathione S-transferase conjugation of 1-chloro-2,4-dinitrobenzene
(Habig and Jakoby, 1981). Each microsomal fraction was tested with 11 additional assays performed in duplicate. P450, cytochrome
b5, and absorbance at 420 contents were
determined from difference spectra (Omura and Sato, 1964).
p-Nitrophenol hydroxylation (Papac and Franklin, 1988), NADH
cytochrome b5 reductase (Rogers and Strittmatter, 1973), and hydrolysis of p-nitrophenolacetate
(Ashour et al., 1987) were determined colorimetrically. The
glucuronidations of morphine and 1-naphthol were determined by HPLC
(Liu and Franklin, 1984).
) as follows. The
incubation, in a final volume of 200 µl, consisted of 0.4 mg of
microsomal protein, 500 µM substrate, a NADPH-generating system
providing 1 mM NADP, 6 mM glucose 6-phosphate, 0.4 units of
glucose-6-phosphate dehydrogenase, and 75 µl of 0.15 M Tris buffer
with 5 mM magnesium chloride (pH 7.4). The assay was initiated by the
addition of the NADPH-generating system in a 37°C shaking water bath
and terminated after 0, 15, or 30 min by the addition of 25 µl of 3 N
hydrochloric acid. The internal standard was added (25 µl of 0.05 mg/ml of chlorpropamide in buffer), and the mixture was vortexed and
extracted with 2 ml of diethyl ether, vortexed, and centrifuged for 10 min at 600g (IEC model K, size 2; IEC, Needham
Heights, MA). The organic layer was collected, air dried at room
temperature, reconstituted with 150 µl of mobile phase, and vortexed
for 5 min (IKA-Vibrax-VXR). The analysis was performed by HPLC using a
Waters 996 photodiode array detector monitoring 233 nm, a Waters
600 Controller, a Waters 717 plus Autosampler (injecting 25-µl
sample), and a Symmetry C18 column (250 × 4.6 mm, 5 µm; Waters, Milford, MA). The mobile phase consisted of methanol/0.01 M monobasic ammonium phosphate (pH 5.4) (60:40, v/v). The
flow rate was 1 ml/min at room temperature.
Dextromethorphan N- and O-demethylation activity
of HLM was modified from the method of Schmider et al. (1997) as
follows. The incubation, in a final volume of 200 µl, consisted of
0.4 mg of microsomal protein, 250 µM substrate, a NADPH-generating system providing 1 mM NADP, 6 mM glucose 6-phosphate, 0.4 units of
glucose-6-phosphate dehydrogenase, and 75 µl of 0.15 M Tris buffer
with 5 mM magnesium chloride (pH 7.4). The assay was initiated by the
addition of the NADPH-generating system in a 37°C shaking water bath
and terminated after 0, 15, or 30 min by the addition of 50 µl of
30% trichloroacetic acid. The internal standard was added (50 µl of
6 µg/ml of levallorphan in buffer), and the mixture was vortexed and
centrifuged for 10 min at 930g (IEC model K, size 2).
Analysis of the supernatant was performed by HPLC using a Waters 474 scanning fluorescence detector monitoring 280-nm excitation and 310-nm
emission wavelengths, a Waters 600 Controller, a Waters 717 plus
Autosampler (injecting 15-µl sample), and a Luna
C18(2) column (150 × 4.6 mm, 5 µm;
Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile
containing 0.06% triethylamine/0.05 M potassium phosphate (pH 2.8)
containing 0.06% triethylamine (28:72, v/v). The flow rate was 1 ml/min at room temperature.
Determination of Kinetic Parameters.
Dextromethorphan O-demethylation was determined as described
above, except that the substrate concentration was varied from 1 to
1500 µM. For each treatment group compared, each substrate concentration was performed in duplicate. LAAM
N-demethylation to norLAAM and dinorLAAM was measured for
substrate concentrations ranging from 0.3 to 1000 µM using our
previously described gas chromatography/mass spectrometric method
(Moody et al., 1995).
was used to
fit the data where n was determined from plots of log
[v/Vmax v]
versus log S. For the latter, the
Vmax was initially estimated from a graph of S versus v, with reiterative determinations of
n as the Vmax was approached
with the sigmoidal equation that was determined using the nonlinear
regression function of KaleidaGraph (version 3.09, Synergy Software,
Reading, PA). For concentrations of LAAM less than 100 µM, linear
results were found in Eadie-Hofstee plots. Vmax and Km
values were determined from the y-axis intercept and the
negative of the slope of linear regression analysis of these plots
(Cricket Graph 1.3, Cricket Software, Malvern, PA). The O-demethylation of dextromethorphan to dextrorphan was
biphasic in Eadie-Hofstee plots. Vmax and
Km values were determined using the
nonlinear regression function of Systat (version 5.2.1, Statistical Solutions, Saugus, MA) to solve the following equation:
v = [(Vmax1)(1 + Km1/S)
1] + [(Vmax2)(1 + Km2/S)1].
Data Analysis and Statistics.
The microsomal data for 12 of the assays were considered positive
indicators of microsomal purity: protein content, NADPH cytochrome P450
reductase, cytochrome P450, NADH cytochrome
b5 reductase, cytochrome
b5, p-nitrophenol hydroxylation,
tolbutamide hydroxylation, dextromethorphan N- and
O-demethylation, glucuronidation of morphine and 1-naphthol,
and ester cleavage of p-nitrophenolacetate. The microsomal
data for three of the assays were considered negative indicators of
microsomal purity: absorbance at 420, succinate dehydrogenase, and
glutathione S-transferase. For each experiment, the 15 indicators were expressed for recovery (per gram of liver), specific
activity (per mg of protein), and for the ratio of activity to
cytochrome P450 reductase activity. For each expression system, the
activities (or contents) were ranked across an experimental group
(e.g., 1, 2, 3 for the highest to lowest activities of positive indicators versus the 6, 8, and 10 strokes). To be ranked differently, the activities must differ by at least 5%. For the two-variable sucrose in homogenate experiments, ties were ranked as 1.5, 1.5. For
the three-variable experiments, the tie rankings were established such
that a difference of 1 would exist between groups and the total of all
three rankings was always 6. Therefore when the two highest activities
were tied, a ranking of 1.7, 1.7, 2.7 was assigned. When the two lowest
activities were tied, a ranking of 1.3, 2.3, 2.3 was assigned. When the
middle activity did not differ from the others by 5% but the extremes
did, a ranking of 1.5, 2.0, 2.5 was assigned. Once rankings were
assigned, experimental groups were then compared using one-way analysis
of variance (p < 0.05) with the Tukey
post hoc test (p < 0.05) (Zar, 1984).
) was used to compare
the substrate concentration-dependent experiments with LAAM and dextromethorphan. Activities determined at any one concentration for
the compared groups from a human liver were treated as matched pairs
(e.g., the activity determined at 100 µM dextromethorphan for liver
CO 06 prepared without sucrose was a matched pair to the activity
determined at 100 µM dextromethorphan for liver CO 06 prepared with
sucrose). To permit comparison between different concentrations, the
percentage of difference between matched pairs was determined and used
as the variable as previously described (Moody et al., 1999).
Therefore, a comparison could be made between microsome preparations at
different substrate concentrations using all three livers for each
comparison. Combining the data in this manner increased the power of
our test.
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Results and Discussion |
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Top
Abstract
Introduction
Results and Discussion
References
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Sedimentation Profiles.
The separation of organelle marker enzymes was initially evaluated
using the qualitative plots first used by de Duve and coworkers (1955)
to demonstrate patterns of enzyme distribution following differential
centrifugation. The specific activities (per mg of protein) of
mitochondrial succinate dehydrogenase, endoplasmic reticular NADPH
cytochrome P450 reductase, and cytosolic glutathione S-transferase were plotted against the percentage of total
protein recovered in the initial pellet, the microsomal pellet, and the cytosol (Fig. 1). In this manner, not
only is the specific activity displayed, but the area for each fraction
is proportional to the percentage of activity recovered. Increasing the
number of homogenization strokes appears to release more cytosolic
material from the pellets, but it decreases specific activity of the
reductase in the microsomes. In the force experiment, the specific
activity and recovery (per gram of liver) in the microsomal and
cytosolic fractions appear to be optimal at 9,000g. In the
homogenization buffer experiment, the addition of sucrose presents a
tradeoff. The sucrose buffer appears to decrease both NADPH cytochrome
P450 reductase specific activity and recovery in the microsomal
fraction but also decreases succinate dehydrogenase contamination in
the microsomal fraction (Fig. 1).
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Recovery and Specific Activity in Microsomes. When direct comparison (one-way analysis of variance) of preparation variables was made between any one of the 15 assays performed, no significant differences were found (data not shown). This was due greatly to the large variation between any three liver samples. To statistically evaluate the different treatments, a method was needed to group the different assays to increase the power of the test. We therefore used a ranking scheme that allowed us to group the results of all the assays and each liver per assay. An example of the ranking scheme is provided in Table 3. A summary of the statistical results is provided in Table 4. In the force experiment, both optimal recovery and specific activity (or specific content) increased significantly with decreasing force. The addition of sucrose to the homogenization buffer significantly increased the relative microsomal specific activity (ratio of activity or content to NADPH cytochrome P450 reductase activity). In the strokes experiment, the relative specific microsomal activity was positively influenced by an increasing number of strokes.
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Kinetics in Different Microsome Preparations.
As a further test of the impact of the microsomal preparation method on
drug-metabolizing enzymes, detailed substrate concentration-dependent experiments were performed using a P450 2D6-specific pathway, dextromethorphan O-demethylation (Kupfer et al., 1984;
Schmider et al., 1997), and a P450 3A4-specific pathway, LAAM
N-demethylation to norLAAM and dinorLAAM (Moody et al.,
1997). Dextromethorphan O-demethylation was compared in
microsomes from livers prepared in homogenization buffer that did, or
did not, contain sucrose. LAAM N-demethylation to norLAAM
and dinorLAAM was compared in microsomes from liver homogenates that
were subjected to initial centrifugation at 9,000 or
12,000g.
), dextromethorphan O-demethylation displayed biphasic
properties in Eadie-Hofstee plots (Fig.
2), and kinetic parameters could be
estimated using nonlinear regression (Table
5). For livers CO 07 and CO 08 the
high-affinity (Km1) and low-affinity
(Km2) components of the biphasic equation
for dextromethorphan O-demethylation were similar (Table 5).
While the Km1 for liver CO 06 was in the
same range, its Km2 was much higher. The
possibility that a third enzyme with intermediate affinity may have
been involved in this liver preparation could not be adequately
approached with the software available, but could represent an
explanation for this extreme variation. No readily apparent differences
in kinetic parameters were noted for livers prepared with sucrose
versus without sucrose in the homogenization buffer (Table 5).
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). The kinetics of LAAM N-demethylation,
however, have not yet been studied in HLM. Because the previous data
suggest that both LAAM and norLAAM are P450 3A4 substrates, we analyzed the combined formation of both products, norLAAM and dinorLAAM. Over a
substrate concentration range of 0.3 to 1000 µM, nonhyperbolic curves
were found for product formation (Fig.
3A). These curves were amenable to
fitting with a sigmoidal Vmax equation that
permitted an initial estimate of kinetic parameters and a comparison in livers prepared using different centrifugation forces (Table
6). While the
Vmax values for livers CO 04 and CO 05 were
similar, a much higher Vmax was estimated
for liver CO 03. The estimated S50 values
were different for each of the three livers. No readily apparent
differences between livers prepared using either 9,000 or
12,000g centrifugation forces were noted.
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),
further studies are needed to ascertain the mechanism for these
kinetics with LAAM metabolism before a reasonable choice can be made.
The nonhyperbolic substrate versus activity curves for
N-demethylation of LAAM may have arisen from a number of
factors that could include substrate inhibition, product inhibition,
involvement of other P450s, or the involvement of other metabolic
pathways (e.g., ester hydrolysis, ring hydroxylation). Our initial
studies on P450 3A4 involvement focused on lower substrate
concentrations that were of therapeutic relevance. When we restrict the
analysis to substrate concentrations less than 100 µM, the curves
approach linearity in Eadie-Hofstee plots (Fig. 3B). Under these
limitations, the Vmax values for livers CO
03, CO 04, and CO 05 prepared using 12,000g force were 420, 225, and 101 (pmol)(min)1(mg of
protein)1, respectively, with corresponding
Km values of 10.0, 10.7, and 45.6 µM. As
it would be highly unlikely for plasma, or even liver, concentrations
of LAAM to exceed 100 µM in even a toxic situation, these latter
parameters may prove more useful for in vitro-in vivo modeling.
A statistical comparison of kinetic parameters, particularly when
biphasic kinetics are present, is a complex matter, with no readily
available solutions. We therefore took the approach that incubations
with the same substrate at the same concentration represented matched
pairs, and used Student's paired t test for evaluation
(Zar, 1984). As an initial test of this statistical approach, we first
compared all of the replicate results for the dextromethorphan
O-demethylations with the first replicate being compared
with the second replicate. As expected, there was no significant
difference between the replicates (Table
7). No significant difference was found
in either dextromethorphan O-demethylation activities in
microsomes prepared with homogenates with or without sucrose or in LAAM
N-demethylation activities from microsomes prepared with the
initial centrifugation at 9,000 versus 12,000g (Table 7).
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Conclusions.
HLM are used widely to characterize the role of P450s and other enzymes
in xenobiotic metabolism. There are, however, many procedural variables
associated with the preparation of microsomes. We examined three of
these variables to better understand how preparatory differences might
affect microsomal purity and the comparison of microsomal data between
research laboratories. The three variables chosen, centrifugation
force, presence of sucrose in the homogenization buffer, and
homogenization strokes, are among those that can have the largest
impact on differential centrifugation (de Duve, 1971) and that have
shown the widest variation between laboratories (Table 1). Although
none of our experiments indicated a significant difference between
procedures by all three measures of microsomal purity and recovery,
three trends did develop.
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Acknowledgments |
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We thank Michael Franklin, University of Utah, for performing the glucuronidation assays, and Kent Kunze, University of Washington, for assistance in evaluating the biphasic kinetic data.
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Footnotes |
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Received November 9, 2000; accepted December 5, 2000.
1 Current Address: Secor International, Inc., Salt Lake City, UT 84107.
This research was supported by U.S. Public Health Service Grant R01 DA10100. A preliminary report of these results was presented at the Experimental Biology 1999 meeting in Washington, D.C.
Send reprint requests to: David E. Moody, Ph.D., University of Utah, Center for Human Toxicology, 20 S. 2030 E. RM 490, Salt Lake City, UT 84112-9457. E-mail: dmoody{at}alanine.pharm.utah.edu
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Abbreviations |
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Abbreviations used are:
HLM, human liver
microsomes;
P450, cytochrome P450;
LAAM, l--acetylmethadol;
HPLC, high-performance liquid
chromatography.
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References |
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Top
Abstract
Introduction
Results and Discussion
References
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-acetylmethadol (LAAM), norLAAM, and methadone.
Drug Metab Dispos
25:
1347-1353-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.
J Anal Toxicol
19:
343-351[Medline].20°C.
J Anal Toxicol
23:
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), 8 strokes (- - -), and 10 strokes (- · -); for the force of first centrifugation experiment:
9,000g (
100 µM. The
microsomes were from liver CO 03 prepared with an initial
centrifugation force of 9000g. Values are the result of
single incubations.