Transiently altered acetaminophen metabolism after liver transplantation.

Article abstract of DOI:10.1016/S0009-9236(03)00062-6

BACKGROUND AND OBJECTIVES: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in drug metabolism occur after organ transplantation. Th

Full text of DOI:10.1016/S0009-9236(03)00062-6

Transiently altered acetaminophen
metabolism after liver transplantation
Background and Objectives: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone
imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in
drug metabolism occur after organ transplantation. This study was designed to characterize acetaminophen
disposition during the first 6 months after liver transplantation.
Methods: Thirteen liver transplant patients received an oral dose of acetaminophen (500 mg) on days 2, 10,
90, and 180 after transplantation. Serial blood samples were collected for 8 hours, and urine was collected for
24 hours. Liver biopsy specimens were obtained from the donor liver during transplantation (day 0) and on
days 10, 90, and 180 after transplantation.
Results: There were significant time-dependent changes in acetaminophen metabolism after liver transplan-
tation. When day 2 and day 10 were compared with day 180, the respective mean urinary recovery was 137%
and 81% higher for thioether conjugates derived from NAPQI (P ؍ .0002 and P ؍ .01, respectively); 31% and
22% lower for acetaminophen sulfate (P ؍ .0006 and P ؍ .008, respectively); and 22% and 27% lower for
acetaminophen glucuronide (P ؍ .05 and P ؍ .004, respectively). Metabolite formation clearances changed
in concordance with the fractional urinary recovery. It was surprising that hepatic CYP2E1 content on day 10
after transplantation was only 20% higher, on average, than that found on day 180 (not significant). In
contrast, hepatic CYP3A4 content was 984% higher, on average, when tissue from days 10 and 180 was
compared after transplantation (P ؍ .007).
Conclusions: Increased recovery of acetaminophen thioether conjugates during the first 10 days after liver
transplantation was a result of impaired glucuronidation and sulfation and enhanced NAPQI formation.
(Clin Pharmacol Ther 2003;73:545-53.)
Jeong M. Park, PharmD, Yvonne S. Lin, PhD, Justina C. Calamia, BS,
Kenneth E. Thummel, PhD, John T. Slattery, PhD, Thomas F. Kalhorn, BS,
Robert L. Carithers, Jr, MD, Adam E. Levy, MD, Christopher L. Marsh, MD, and
Mary F. Hebert, PharmD Seattle, Wash
Hepatic glucuronidation and sulfation are the major
pathways of acetaminophen (INN, paracetamol) elimi-
nation, accounting for approximately 50% and 35% of
a therapeutic dose, respectively.1 Less than 10% of a
therapeutic dose is oxidized to N-acetyl-p-
benzoquinone imine (NAPQI), a highly reactive qui-
none imine that is rapidly detoxified by conjugation
with glutathione and eventually excreted into urine as
thioether metabolites. In situations involving acetamin-
ophen overdose, which saturates the sulfation path-
way,2,3 or induction of the oxidative pathway by etha-
nol,4 a greater fraction of the acetaminophen dose is
converted to NAPQI. Excessive NAPQI formation can
result in glutathione depletion and hepatotoxicity.5
Recently, Burckart et al6 reported that oral chlor-
zoxazone clearance, a selective in vivo probe of hepatic
cytochrome P450 (CYP) 2E1 metabolic activity,7-9 was
increased perioperatively among patients undergoing
orthotopic liver transplantation (OLT). Because acet-
aminophen is commonly recommended to this popula-
tion for analgesia or fever reduction and because
CYP2E1 is the principal catalyst for NAPQI formation
in vivo in humans,10-13 we assessed whether NAPQI
formation is increased after OLT. Given the fluctuating
From the Departments of Pharmacy, Pharmaceutics, Medicine, Di-
vision of Hepatology, and Surgery, University of Washington.
Supported by National Institutes of Health grants GM32165 and
M01-RR-00037.
Received for publication Sept 4, 2002; accepted Feb 21, 2003.
Reprint requests: Mary F. Hebert, PharmD, Department of Pharmacy,
University of Washington, H-375 Health Sciences Center, Box
357630, Seattle, WA 98195-7630.
E-mail: mhebert@u.washington.edu
Copyright © 2003 by the American Society for Clinical Pharmacol-
ogy & Therapeutics.
0009-9236/2003/$30.00 ϩ 0
doi:10.1016/S009-9236(03)00062-6
545

CLINICAL PHARMACOLOGY & THERAPEUTICS
JUNE 2003
546 Park et al
nature of hepatic function and immunosuppressive reg-
imens in this population, we studied OLT patients on
multiple occasions during the first 6-month postopera-
tive period. To verify the mechanistic basis for induc-
tion of the NAPQI formation pathway, we measured
total CYP2E1 content in liver biopsy tissue collected,
per clinical protocol, during the same 6-month period.
Hepatic CYP3A4 content was also measured, as it is
often the dominant hepatic P450 isozyme,14 is induc-
ible by some immunosuppressive drugs,15,16 and can
catalyze NAPQI formation.10,17
closed for at least 48 hours after each dose of acetamin-
ophen.
All 15 study subjects received a standard posttrans-
plantation immunosuppressive regimen. Of these, 12
received a quadruple immunosuppressive regimen (cy-
closporine [INN, ciclosporin], azathioprine, pred-
nisone, and daclizumab induction therapy). For the
other 3, azathioprine was omitted from the regimen of
2 subjects, because of preexisting hepatocellular carci-
noma in the cirrhotic organ, and 1 subject received
tacrolimus and sirolimus instead of cyclosporine as part
of the maintenance regimen.
METHODS
Standards and analytic reagents
Study design
Synthetic unlabeled acetaminophen metabolite stan-
dards (glucuronide, sulfate, and S-methyl) were kindly
provided by Dr Sid Nelson, Department of Medicinal
Chemistry, University of Washington, Seattle, Wash.
Other unlabeled thioether metabolites were isolated
from mouse urine, as described below. Unlabeled acet-
aminophen was purchased from Sigma Chemical Co
Acetaminophen, as a 500-mg oral tablet (McNeil
Consumer Products Co, Fort Washington, Pa) or
500-mg oral solution (Roxane Laboratories, Inc, Co-
lumbus, Ohio), was given to each subject on four oc-
casions: approximately 2, 10, 90, and 180 days after
transplantation. On each study day, serial blood sam-
ples were collected at 0, 0.5, 3, and 8 hours after
administration of the acetaminophen dose. Blood-
sampling frequency was limited for patient safety con-
siderations during the early perioperative period.
Plasma was separated from blood cells by centrifuga-
tion and stored at Ϫ80°C until analyzed for acetamin-
ophen. Urine was collected for 24 hours after acetamin-
ophen ingestion. It was stabilized with 3 g of ascorbic
acid and maintained at 4°C during the collection period.
Total 24-hour urine volume was recorded and an ali-
quot stored at Ϫ80°C until analyzed for acetaminophen
and its metabolites.
A core needle biopsy specimen of the liver was
collected according to clinical protocol during the
transplantation operation (day 0) and approximately 48
hours after the acetaminophen kinetic experiments on
days 10, 90, and 180. When available, approximately
10 to 30 mg of each biopsy specimen in excess to that
needed for diagnostic purposes was used for research.
The biopsy tissue was placed in 100 to 300 ␮L of
homogenization buffer (10 mmol/L potassium phos-
phate, pH 7.4; 0.25 mol/L sucrose; Complete protease
inhibitor cocktail with ethylenediaminetetraacetic acid
[Boehringer Mannheim, Germany]), frozen immedi-
ately on dry ice, and stored at Ϫ80°C until Western blot
analysis was performed. Just before analysis, the tissue/
buffer mixture was thawed on ice and homogenized
with the use of a 1-mL glass pestle-glass homogeniza-
tion tube. Approximately 20 complete strokes were
applied by hand.
2
(St Louis, Mo). H3-Labeled acetaminophen was syn-
thesized as follows. In brief, 2.9 g of p-aminophenol
(Aldrich, Milwaukee, Wis) was stirred into 100 mL of
acetonitrile in a 200-mL round-bottom flask. To this
was added 3.0 mL of deuterium-labeled 2H6-acetic
anhydride (Aldrich). The flask was sealed and stirred
overnight in the dark at room temperature. The result-
ing crystalline slurry was chilled to Ϫ20°C for 2 hours,
and acetaminophen crystals of high purity were isolated
by filtration. The recovered molar yield was 60%. Iso-
topic purity of the final product was assessed by mass
spectrometry and found to consist of a 100:5:Ͻ0.1
mole ratio of 2H3:2H2:2H1-acetaminophen. Male Swiss-
Webster mice, weighing 25 to 35 g, were purchased
from Animal Tech (Kent, Wash). ␤-Glucuronidase/
sulfatase enzyme, a crude extract from Helix pomatia,
was purchased from Sigma Chemical Co. All other
reagents were of analytic grade or better.
Human subjects
This study was approved by the Institutional Review
Board at the University of Washington, Seattle, Wash,
and written informed consent was obtained from all
subjects. Fifteen patients receiving an OLT at the Uni-
versity of Washington Medical Center (UWMC) were
enrolled. Liver transplantation operations were per-
formed according to standard protocols for the UWMC
Department of Transplant Surgery. The common bile
duct was anastamosed during surgery with or without a
T tube. In subjects with a T tube, the external port was

CLINICAL PHARMACOLOGY & THERAPEUTICS
VOLUME 73, NUMBER 6
Park et al 547
Quantitation of acetaminophen and metabolites
each metabolite. For measurement of acetaminophen
and its metabolites in urine, the respective interday and
intraday variability (coefficient of variation for repeated
measures) was as follows: glucuronide, 11.1% and
0.9%; sulfate, 5.7% and 1.2%; S-methyl, 14.3% and
7.9%; cysteine, 13.9% and 3.2%; mercapturate, 7.4%
and 4.0%; and acetaminophen, 3.4% and 2.2%. Limits
of quantitation were not rigorously assessed. All mea-
sured urine metabolite concentrations were above that
of the lowest standard: glucuronide, 17 ␮mol/L; sulfate,
19 ␮mol/L; S-methyl, 0.8 ␮mol/L; cysteine, 3 ␮mol/L;
and mercapturate, 7.5 ␮mol/L.
Plasma acetaminophen concentration and acetamin-
ophen sulfate and glucuronide concentrations in unhy-
drolyzed urine were quantified by HPLC, as described
previously18 with minor modifications. The concentra-
tion of acetaminophen thioether metabolites derived
from NAPQI conjugation with glutathione (3-cysteinyl,
3-mercapturic acid, and 3-S-methyl) in hydrolyzed
urine (␤-glucuronidase/sulfatase treated for 24 hours at
37°C) was quantified by liquid chromatography-mass
spectrometry. Because of the instability of 3-cysteinyl
and 3-mercapturic acid metabolites with prolonged
storage, fresh metabolites were isolated by HPLC (re-
peated injections of urine from a mouse and human
dosed with acetaminophen), just before the analysis of
study samples, following the general elution method
described by Chien et al.18 Synthetic 3-S-methyl me-
tabolite was repurified by the same HPLC method.
Metabolite concentrations in the purified stock solution
were determined by comparison of the ultraviolet ab-
sorbance at 254 nm for metabolites and an acetamino-
phen stock solution of known concentration, with cor-
rection for differences in the molar absorptivity
coefficients of the metabolites and acetaminophen.
Deuterated internal standards for the thioether me-
tabolites were generated biologically. In brief, 200
mg/kg 2H3-labeled acetaminophen was administered
intraperitoneally to Swiss-Webster mice. Urine (0-12
hours) was collected, diluted 1:5 with 12.5 mmol/L
sodium acetate (pH 5.0), aliquoted, and stored at
Ϫ80°C. Analysis for thioether metabolite concentra-
tions in the hydrolyzed urine of study subjects was
performed on an Agilent Technologies 1100 Series
Liquid Chromatograph/Mass Selective Detector (Palo
Also, Calif), operated in the positive ion electrospray
mode with selective ion monitoring. A Phenomenex
Luna 3-␮m, C8 (100 ϫ 2-mm) column (Phenomenex
Inc, Torrance, Calif) was used for nominal chromato-
graphic separation. The mobile phase was 20 mmol/L
acetic acid (pH 3.5) delivered at a flow rate of 0.22
mL/min. The following mass spectrometry conditions
were used: capillary and fragmentor voltages of 3300
and 100 V, respectively, and nitrogen (N2) as a drying
gas at a flow rate of 10 L/min, a temperature of 300°C,
and a nebulizer gas pressure of 25 psi. The following
pairs of ions (unlabeled and deuterated internal stan-
dard) were detected by selective ion monitoring: mass-
to-change ratio (m/z) 198 and 201 (3-S-methyl), m/z
271 and 274 (3-cysteinyl), and m/z 313 and 316 (3-
mercapturic acid).
Pharmacokinetic analysis
The area under the acetaminophen plasma
concentration-time curve (AUC) for the 0- to 8-hour
interval was calculated with the use of the log-linear
trapezoidal rule. The AUC from 8 hours to infinity was
calculated as the ratio C8 hours/ke, in which C8 hours is the
acetaminophen plasma concentration at 8 hours after
dosing and ke is the terminal elimination rate constant
obtained from the 3- and 8-hour plasma concentration
data. Apparent oral clearance (CL/F) was calculated as
dose/total AUC. The molar amount of each thioether
metabolite (3-mercapturate, 3-cysteinyl, and 3-S-
methyl) recovered in urine was summed and the total
divided by the acetaminophen dose for calculation of
the fraction of acetaminophen dose metabolized to
NAPQI. The fraction of dose metabolized to acetamin-
ophen glucuronide and acetaminophen sulfate was cal-
culated in an analogous manner.
Western blot analysis
Western blot analysis for CYP2E1 content in indi-
vidual liver homogenate samples was performed with
the use of specific goat polyclonal antihuman CYP2E1
serum (Daiichi Pure Chemical Co, Tokyo, Japan) as the
primary antibody and antigoat immunoglobulin G al-
kaline phosphatase conjugate (Sigma Chemical Co) as
the secondary antibody. CYP2E1 protein levels were
quantified by densitometry, with purified human
CYP2E1 used as a reference standard.19 CYP3A4 con-
tent in liver homogenates was determined by a similar
method, as described previously.20
Statistical analysis
One-way repeated-measures ANOVA, followed by
paired comparisons testing, was conducted to evaluate
pharmacokinetic and enzyme expression differences
among the four study days. Data are reported as mean
Ϯ SD.
Quantitation was performed by comparison of peak
area ratios for unknown samples to standard curves for

CLINICAL PHARMACOLOGY & THERAPEUTICS
JUNE 2003
548 Park et al
Table I. Study subject demographics and relevant drug therapy
Subject Age
No.
(y)
Sex
Ethnicity
Liver disease
Hepatitis C
Relevant concomitant medications*
Clotrimazole (d 10, 90), omeprazole (d 90)
L01
L02
60
48
F
M
White
White
Hepatitis C/ethanol Ranitidine (d 2), clotrimazole (d 10), ciprofloxacin (d
180)
L03
L04
L05
64
52
53
M
M
F
African American Hepatitis C
Clotrimazole (d 10, 90), ranitidine (d 90)
White
White
Hepatitis C/ethanol Clotrimazole (d 2, 10), ranitidine (d 10, 90)
Hepatitis C
Ethanol
Dapsone (d 10, 90, 180), clotrimazole (d 10), omeprazole
(d 10, 90, 180)
L06
L07
51
57
M
F
White
White
Vancomycin (d 2), ranitidine (d 2), clotrimazole (d 10),
nefazodone (d 10, 90), insulin (d 180)
Levothyroxine (d 2, 10), clotrimazole (d 10), ranitidine (d
10), omeprazole (d 90), ursodiol (INN, ursodeoxycholic
acid) (d 90)
␣1-Antitrypsin
deficiency
L08
L09
L10
L11
L12
L13
L14
L15
54
57
49
47
48
50
44
47
M
M
M
M
F
White
Hispanic-Latino
White
Hepatitis C
Clotrimazole (d 10), insulin (d 10, 90, 180), ranitidine (d
10, 90)
Primary sclerosing Clotrimazole (d 2, 10), ranitidine (d 2, 10, 90, 180),
cholangitis
Hepatitis C
insulin (d 180)
Clotrimazole (d 2, 10), ranitidine (d 2, 10), insulin (d
180), DHEA (d 180)
Metoclopramide (d 2, 10), clotrimazole (d 10, 90),
ranitidine (d 10)
Omeprazole (d 2, 10, 90), clotrimazole (d 10), insulin (d
10), vitamin D (d 90, 180)
Dapsone (d 2, 10), clotrimazole (d 10, 90), ranitidine (d
10, 90), insulin (d 10, 90, 180), ursodiol (d 90, 180)
Ranitidine (d 2), clotrimazole (d 10), fluoxetine (d 10, 90,
180)
Clotrimazole (d 2, 10, 90), paroxetine (d 90, 180), insulin
(d 90, 180)
White
Hepatitis C
Hepatitis C
Hepatitis C
White
M
F
White
White
Hereditary
telangiectasia
Hepatitis C
M
White
DHEA, Dehydroepiandrosterone.
*List of concomitant medications that might alter hepatic P450 or uridine diphosphate–glucuronosyltransferase expression and function and acetaminophen
metabolism, in addition to standard immunosuppressive or prophylaxis therapies described in Methods section. Study dates in parentheses indicate concomitant drug
therapy on day of acetaminophen administration.
RESULTS
study data for another subject were excluded from
statistical analysis because of apparent acetaminophen
use immediately before the study.
The majority of study subjects were white men aged
approximately 50 years. Additional demographic and
clinical information is presented in Table I. The major
cause of liver disease in study subjects was hepatitis C
infection. Recurrent infection after transplantation is
common and might have affected hepatic enzyme ex-
pression. However, there was no evidence that this
occurred on the basis of the acetaminophen metabolic
profile for the study group. Similarly, we identified a
number of concomitant drugs that might alter acetamin-
ophen metabolism (Table I) but found no evidence for
an association between use of these drugs and urine
metabolite recovery.
The apparent total body clearance (CL/F) and half-
life of acetaminophen varied significantly over the 180-
day posttransplantation study period (P ϭ .02 and
.0007 by ANOVA, respectively) (Table II). Moreover,
mean clearance and half-life parameters for day 180
were similar to those we have reported previously for
healthy volunteers.4,18 On the basis of these findings,
postoperative day 180 was chosen as a reference time
point for longitudinal comparisons of urinary recovery
and formation clearance data from subjects.
The urinary recovery of thioether conjugates varied
significantly over the 6-month study period: 15.4% Ϯ
4.3%, 12.5% Ϯ 5.2%, 6.3% Ϯ 2.8%, and 7.7% Ϯ 3.5%
for postoperative days 2, 10, 90, and 180, respectively
(P Ͻ .0001 by ANOVA) (Table III). These parameters
Thirteen of fifteen enrolled subjects completed all
four of the pharmacokinetic study days. One subject
discontinued study participation after the day 90 study
because of the need for retransplantation. Day 180

CLINICAL PHARMACOLOGY & THERAPEUTICS
VOLUME 73, NUMBER 6
Park et al 549
Table II. Acetaminophen pharmacokinetic parameters
Parameter
Day 2
Day 10
Day 90
Day 180
CL/F (mL/h)
t1/2 (h)
19.6 Ϯ 6.41 (.07)
4.50 Ϯ 2.46 (0.02)
28.4 Ϯ 6.77 (NS)
2.19 Ϯ 0.35 (.02)
26.5 Ϯ 7.24 (NS)
2.34 Ϯ 0.48 (.001)
24.2 Ϯ 7.84
2.62 Ϯ 0.47
Data are presented as mean Ϯ SD, with P values in parentheses representing paired comparisons for day 2, 10, or 90 versus day 180. For ANOVA, P ϭ .02 for CL/F
and P ϭ .0007 for t1/2
.
CL/F, Apparent oral clearance; t1/2, half-life; NS, not significant.
Table III. Urinary recovery and formation clearances for acetaminophen metabolites
Parameter
Day 2
Day 10
Day 90
Day 180
Urinary recovery (% dose)*
Thioether conjugates
Sulfate
15.4 Ϯ 4.29 (.0002)
27.2 Ϯ 7.94 (.0006)
27.7 Ϯ 14.7 (.05)
12.2 Ϯ 5.18 (.01)
30.1 Ϯ 6.82 (.008)
26.8 Ϯ 8.55 (.004)
6.30 Ϯ 2.81 (NS)
37.7 Ϯ 9.00 (NS)
34.7 Ϯ 9.08 (NS)
7.67 Ϯ 3.49
40.6 Ϯ 9.66
38.5 Ϯ 9.71
Glucuronide
Formation clearance (L/h)†
Thioether conjugates
Sulfate
2.87 Ϯ 0.90 (.004)
5.14 Ϯ 1.96 (.0006)
5.67 Ϯ 3.53 (.02)
3.33 Ϯ 1.30 (.0008)
8.50 Ϯ 2.46 (NS)
7.95 Ϯ 4.15 (NS)
1.59 Ϯ 0.61 (NS)
9.87 Ϯ 3.05 (NS)
9.36 Ϯ 3.82 (NS)
1.78 Ϯ 0.81
9.69 Ϯ 3.60
9.56 Ϯ 4.37
Glucuronide
Data are presented as mean Ϯ SD, with P values in parentheses representing paired comparisons for day 2, 10, or 90 versus day 180.
*For ANOVA, P Ͻ .0001 for thioether conjugates, P ϭ .0004 for sulfate, and P ϭ .02 for glucuronide.
†For ANOVA, P Ͻ .0001, P ϭ .0003, and P ϭ .06, respectively.
represent the minimum fraction of the acetaminophen
dose converted to NAPQI. Compared with day 180,
thioether recovery was, on average, 137% and 81%
higher on days 2 and 10 after transplantation, respec-
tively (P ϭ .0002 and .01, respectively) (Table III, Fig
1). Interestingly, for one individual (subject L14),
thioether conjugate recovery remained elevated (ap-
proximately 13% of the dose) over the entire 180-day
study period (Fig 1).
The urinary recovery of acetaminophen sulfate also
varied significantly over the 6-month study period:
27.2% Ϯ 7.9%, 30.1% Ϯ 6.8%, 37.7% Ϯ 9.0%, and
40.6% Ϯ 9.7% for postoperative days 2, 10, 90, and
180, respectively (P ϭ .0004 by ANOVA) (Table III).
Compared with day 180, acetaminophen sulfate recov-
ery was, on average, 31% and 22% lower on days 2 and
10 after transplantation, respectively (P ϭ .0006 and
.008, respectively) (Table III). There were also differ-
ences in the mean urinary recovery of acetaminophen
glucuronide over the 6-month study period: 27.7% Ϯ
14.7%, 26.7% Ϯ 8.6%, 34.7% Ϯ 9.1%, and 38.5 Ϯ
9.7% for postoperative days 2, 10, 90, and 180, respec-
tively (P ϭ .02). Compared with day 180, acetamino-
phen glucuronide recovery was, on average, 22% and
27% lower on days 2 and 10 after transplantation,
respectively (P ϭ .05 and .004, respectively) (Table
III).
Fig 1. Time course of change in fraction of acetaminophen
dose converted to N-acetyl-p-benzoquinone imine (NAPQI).
Each data point represents the sum of all acetaminophen
thioether metabolites recovered in urine divided by the ad-
ministered dose. The mean urinary recovery from 13 subjects
for each time period is shown as solid triangles.
(calculated as the product of clearance and urinary
recovery fraction) varied significantly during the first
180 days after transplantation (P Ͻ .0001, P ϭ .0003,
and P ϭ .06 by ANOVA, respectively) (Table III). The
thioether formation clearance was, on average, 97%
and 122% higher on postoperative days 2 and 10,
respectively, compared with day 180 (P ϭ .004 and
.0008, respectively). In contrast, the acetaminophen
sulfate and glucuronide formation clearances were, on
average, 42% and 20% lower on day 2 after transplan-
The formation clearance of thioether conjugates,
acetaminophen sulfate, and acetaminophen glucuronide

CLINICAL PHARMACOLOGY & THERAPEUTICS
JUNE 2003
550 Park et al
Fig 2. CYP2E1 content (A) and CYP3A4 content (B) in liver biopsy specimens from 8 subjects,
as estimated by Western blot immunodetection, during first 6 months after liver transplantation.
Mean values for each posttransplantation period are shown as solid triangles.
tation, respectively, as compared with day 180 after
tion clearance to thioether conjugates for these same 8
subjects was 81% higher on day 10, on average, com-
pared with day 180 after transplantation (P ϭ .006).
In contrast to the generally invariant apparent
CYP2E1 expression, hepatic CYP3A4 levels varied
significantly over the 180-day study period (P ϭ .02 by
ANOVA) (Fig 2, B). Hepatic CYP3A4 content in-
creased several-fold between day 0 and day 10 and
declined gradually back to baseline by day 180 after
transplantation. CYP3A4 content in day 10 biopsy tis-
sue was, on average, 984% higher than that found in the
matched day 180 specimens (P ϭ .007).
transplantation (P ϭ .0006 and .02, respectively) (Ta-
ble III). Acetaminophen sulfate and glucuronide forma-
tion clearances were also lower on postoperative day
10, but the differences compared with day 180 were not
significant.
For 8 of 15 subjects, a portion of liver biopsy spec-
imens collected on days 0, 10, 90, and 180 after trans-
plantation were made available for research. Although
hepatic CYP2E1 content varied between subjects, the
ANOVA analysis indicated no significant change over
the 180-day study period (Fig 2, A). A paired compar-
ison revealed only a 20% higher level of CYP2E1 in the
day 10 biopsy specimen, on average, compared with the
day 180 biopsy specimen. However, the mean forma-
The oxidation of acetaminophen by liver homoge-
nates could not be measured because of insufficient
tissue mass and limited assay sensitivity.

CLINICAL PHARMACOLOGY & THERAPEUTICS
VOLUME 73, NUMBER 6
Park et al 551
Burckart et al6 studied CYP2E1 activity in liver
transplantation patients using the plasma concentration
ratio of 6-hydroxychlorzoxazone/chlorzoxazone at 4
hours after chlorzoxazone administration as an index of
CYP2E1 metabolic activity. They reported a significant
increase in the metabolic ratio in the first month after
liver transplantation and a subsequent decline in the
ratio over time to a mean value similar to that seen in
healthy control subjects. These results are in agreement
with our observed perioperative increase in NAPQI
formation clearance in OLT subjects but are again at
odds with a failure to demonstrate appreciable induc-
tion of hepatic CYP2E1 content. The reason for this
discrepancy is unknown, but our suggestion of a change
in the CYP2E1 holoenzyme/apoprotein pool also ap-
plies. There may also have been significant periopera-
tive changes in chlorzoxazone plasma protein binding
(approximately 97% in healthy volunteers) that could
confound a mechanistic interpretation of the results of
Burckart et al.
DISCUSSION
The mean fraction of a single 500-mg acetaminophen
dose converted to the protoxic metabolite NAPQI was
enhanced by approximately 100% during the first 10
days after liver transplantation. For some OLT patients,
urinary recovery of thioether metabolites represented as
much as 25% of the administered dose. This metabolic
change appeared to be transient, and by 3 to 6 months
after OLT, thioether recovery had returned to a mean
value similar to that which we have reported for healthy
volunteers.4,18 The mean increase in NAPQI formation
observed in this study population was modest but
greater than that reported after short-term ethanol in-
gestion4 and long-term isoniazid treatment,18,21 drugs
for which an increased risk of hepatotoxicity has been
suggested.22-25 Individual thioether conjugate urinary
recovery fractions of 15% to 25% are comparable to
recovery values reported for poisoned patients with
severe liver injury.1-3
Although we had expected to find elevated NAPQI
formation in our subjects, on the basis of published
chlorzoxazone clearance data in OLT patients,6 an in-
crease in hepatic CYP2E1 content during the same
early perioperative period was also anticipated. How-
ever, significant postoperative changes in CYP2E1 con-
tent were not evident (Fig 2, A). Hepatic CYP2E1
content was 20% higher on day 10, as compared with
day 180, but this effect was modest in comparison to
the 81% higher NAPQI formation clearance (day 10
versus day 180) for the same subset of subjects.
CYP2E1 content was not measured at 2 days after
OLT, a time when NAPQI formation was also en-
hanced. It is possible that there was a shift in the ratio
of CYP2E1 holoenzyme (heme present) to apoprotein
(inactive enzyme, heme lost) during the early periop-
erative period, such that the pool of active enzyme
actually increased, whereas there was only a minimal
increase in total CYP2E1 protein detected by Western
blot analysis. Discrimination between the two protein
pools in future studies may help to resolve the discrep-
ancy between posttransplantation changes in protein
content and metabolic activity.
It is unlikely that the observed induction of hepatic
CYP3A4 content during the perioperative period con-
tributed significantly to the enhancement of NAPQI
formation and urine thioether metabolite recovery.
Treatment of healthy volunteers with the potent
CYP3A4 inducer rifampin (INN, rifampicin) failed to
significantly alter NAPQI formation clearance,13 sug-
gesting that the intrinsic clearance for CYP3A4 may be
too low to have a clinical impact under either control or
induced conditions.
In addition to an increase in NAPQI formation clear-
ance, the urinary recovery of thioether metabolites was
elevated by the decline in formation clearances for
acetaminophen conjugation pathways. Acetaminophen
glucuronide and sulfate formation clearances and cor-
responding urinary metabolite recoveries were reduced
on days 2 and 10 after transplantation, as compared
with postoperative day 180. Previous investigators have
reported that the extent of glucuronidation and sulfation
of acetaminophen in clinically stable OLT patients is
similar to that seen in healthy control subjects.26 How-
ever, the time after transplantation was not specified in
the report, and patients may have been studied 3
months or more after OLT.
The biologic basis for a decrease in glucuronidation
and sulfation during the early post-OLT period is not
clear. It is possible that the period of brain death pre-
ceding liver procurement or the liver transplantation
procedure itself resulted in a transient decrease in he-
patic pools of the conjugation enzymes (ie, uridine
diphosphate-glucuronosyltransferase or sulfotrans-
ferase). In addition, it is possible that reduced dietary
intake of inorganic sulfate, expected during and after
surgery, might have reduced cosubstrate availability
(ie, 3'-phosphoadenosine 5'-phosphosulfate) for sulfa-
tion. These homeostatic perturbations may have lasted
for several days and may account for the observed
reduction in acetaminophen conjugation clearances, as
well as enhanced thioether recovery.
Acetaminophen hepatotoxicity is an uncommon
event that, when it occurs, is associated with the inges-
tion of doses above the recommended 4-g maximum

CLINICAL PHARMACOLOGY & THERAPEUTICS
JUNE 2003
552 Park et al
daily dose.2,27 Although we could find no mention in
the literature of liver injury from postoperative use of a
therapeutic dose of acetaminophen in OLT patients,
diagnosis of such an event would be problematic, be-
cause there are other known causes for perioperative
hepatic injury that might obscure such a clinical diag-
nosis. Hepatic function immediately after OLT is inev-
itably compromised to some degree, and in some pa-
tients there can be a significant loss of hepatocytes.
This acute predisposition for liver injury and the en-
hancement of NAPQI formation observed in this study
suggest a brief period of greater risk for acetaminophen
toxicity in OLT patients than in the general population.
In summary, the oxidation of acetaminophen to
NAPQI and urinary recovery of thioether metabolites
was elevated during the first 10 days after OLT. This
change was the result of a reduction in parallel glucu-
ronidation and sulfation clearance pathways and an
increase in NAPQI formation clearance. Accordingly,
the risk of hepatotoxicity from acetaminophen in the
early post-OLT period may be increased, and a reduc-
tion in the maximum daily dose of acetaminophen
during that time should be considered.
8. Kharasch ED, Thummel KE, Mhyre J, Lillibridge JH.
Single-dose disulfiram inhibition of chlorzoxazone me-
tabolism: a clinical probe for P450 2E1. Clin Pharmacol
Ther 1993;53:643-50.
9. Kharasch ED, Hankins DC, Jubert C, Thummel KE,
Taraday JK. Lack of single-dose disulfiram effects on
cytochrome P-450 2C9, 2C19, 2D6, and 3A4 activities:
evidence for specificity toward P-450 2E1. Drug Metab
Dispos 1999;27:717-23.
10. Patten CJ, Ishizaki H, Aoyama T, Lee M, Ning SM,
Huang W, et al. Catalytic properties of the human cyto-
chrome P450 2E1 produced by cDNA expression in
mammalian cells. Arch Biochem Biophys 1992;299:163-
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11. Raucy J, Lasker J, Lieber C, Black M. Acetaminophen
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P450IA2. Arch Biochem Biophys 1989;271:270-83.
12. Sarich T, Kalhorn T, Magee S, al-Sayegh F, Adams S,
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21-8.
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15. Pichard L, Fabre I, Daujat M, Domergue J, Joyeux H,
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A portion of this work was conducted through the Clinical Re-
search Center Facility at the University of Washington. We thank the
clinical staff of the Clinical Research Center and the organ transplant
recovery ward for their excellent assistance in the conduct of this
study.
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