International Journal of Clinical Medicine, 2013, 4, 432-439
http://dx.doi.org/10.4236/ijcm.2013.410078 Published Online October 2013 (http://www.scirp.org/journal/ijcm)
Effects of Cholestasis on Whole Blood Concentration of
Tacrolimus, an Immunosuppressant, in Living-Related
Liver Transplant Recipients*
Shinji Kobuchi1#, Keizo Fukushima2, Yuta Maeda1, Takatoshi Kokuhu3, Hidetaka Ushigome4,
Norio Yoshimura4, Nobuyuki Sugioka2, Kanji Takada1
1Department of Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan; 2Department of Clinical Pharmacokinetics, Fac-
ulty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan; 3Department of Hospital Pharmacy, Kyoto Prefectural Uni-
versity of Medicine, Kyoto, Japan; 4Department of Transplantation and Regenerative Surgery, Kyoto Prefectural University of Medi-
cine, Kyoto, Japan.
Email: #ky05122@poppy.kyoto-phu.ac.jp
Received July 19th, 2013; revised August 20th, 2013; accepted September 15th, 2013
Copyright © 2013 Shinji Kobuchi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: To explore the effects of cholestasis on whole blood concentration of tacrolimus (TAC), an immunosup-
pressant, we investigated the relationship among blood TAC concentration, bile flow, and TAC metabolites in bile, as
well as the relationship between total bilirubin (T-Bil), an index of cholestasis, and blood TAC concentration, in liver
transplant recipients. Methods: Data were collected retrospectively from 16 male and 19 female patients (mean age: 38
years; range: 12 - 59 years) who had undergone a living-related liver transplantation at Kyoto Prefectural University of
Medicine from 2004 through 2008. Analysis of TAC, demethyl-TAC, and hydroxy-TAC in bile was performed by
LC-MS/MS. Results: There was no correlation between the ratio of TAC metabolite to TAC in bile (M/P) of de-
methyl-TAC and post operation days (POD), whereas a weak linear correlation was demonstrated between M/P of hy-
droxy-TAC and POD (r = 0.345). Moreover, linear correlations were not observed between M/P and the TAC trough
level normalized dose (TLTAC/dose), and between TLTAC/dose and POD. A negative linear correlation was demonstrated
between bile flow and T-Bil in blood (r = 0.495). Furthermore, a positive linear correlation was observed between
TLTAC/dose and T-Bil (r = 0.598), whereas there was no correlation between bile flow and TLTAC/dose. Conclusions:
Improvement of hepatic function and the increase of TAC clearance after postoperative day 7 did not significantly con-
tribute to hepatic TAC metabolism, bile excretion, and TLTAC/dose. Postoperative biliary stricture from liver transplan-
tation with/without biliary drainage caused inter- and intra-patient variability in TLTAC/dose after liver transplantation,
which could be assessed by T-Bil. T-Bil in blood might be a predictive biomarker for determining the degree of bile
duct stricture and TAC dose in liver transplantation patients. Along with an appropriate dosing regimen, therapeutic
drug monitoring including T-Bil would be beneficial and enable individual adjustment of TAC dose in liver transplanta-
tion patients.
Keywords: Pharmacokinetics; Bile Duct Stricture; Tacrolimus; Drug Metabolism; Total Bilirubin; Therapeutic Drug
Monitoring
1. Introduction
Tacrolimus (TAC), which is an immunosuppressive
agent (molecular weight of non-hydrate: 806), inhibits
the signal-transduction pathway that leads to T-lympho-
cyte activation [1,2]. TAC is used for preventing or
treating graft rejection after organ transplantation [3],
and is superior to cyclosporine (CyA) in improving sur-
vival (patient and graft) and preventing acute rejection in
living-related liver transplantation [4]. TAC is used as
the first choice drug for living-related liver transplant re-
cipients at our university hospital, Kyoto Prefectural
University of Medicine. Pharmacokinetic parameters of
TAC show high variability, particularly in the early pe-
riod after transplantation, which remains unexplained
[5,6]. TAC has high lipophilicity and insolubility in wa-
*The authors declare that no conflict of interest exists.
#Corresponding author.
Copyright © 2013 SciRes. IJCM
Effects of Cholestasis on Whole Blood Concentration of Tacrolimus, an Immunosuppressant,
in Living-Related Liver Transplant Recipients
433
ter, and is a known substrate both of the drug efflux
pump, P-glycoprotein (Pgp), and metabolizing enzyme,
cytochrome P450 (CYP) 3A [7]. These physicochemical
properties of TAC cause large variations in oral absorp-
tion and extensive metabolism in clearance from the
body. Moreover, a narrow therapeutic window necessi-
tates therapeutic drug monitoring (TDM) to maintain
efficacy of TAC [8,9] and minimize toxicity [10,11].
Liver transplantation is a widely accepted treatment
for end-stage hepatic disease. Despite improvements in
organ preservation technology, surgical technique, and
immunosuppressive strategies, postoperative biliary stric-
ture remains a significant cause of morbidity and mortal-
ity after liver transplantation [12]. Bile duct anastomotic
stricture is a common complication after orthotopic liver
transplantation and has a reported prevalence of 15% -
20% [13]. Previous study found that cholestasis caused
by bile duct ligation induced intestinal bile deficiency
and accumulation of biliary compounds in the body [14].
Therefore, decreased availability of bile salts for solubi-
lization of lipid-soluble drugs following bile duct stric-
ture might decrease intestinal absorption of TAC after
liver transplantation. Moreover, accumulation of bile
acids in liver tissue with resultant free radical production,
hepatic stellate cell activation, and liver fibrosis [15-17]
might decrease hepatic metabolism of TAC in liver
transplantation patients. On the other hand, biliary drain-
age in living-related liver transplantation plays an impor-
tant role in the prevention of biliary anastomotic compli-
cations and the treatment of biliary anastomotic stenosis
after transplantation [18]. However, wide variations in
bile flow and hepatic function in the early stage after
liver transplantation with biliary drainage [12,19] hinder
the control of TAC concentration in TDM.
In biliary drainage at Kyoto Prefectural University of
Medicine, half of the bile excreted from the drain is re-
turned to the intestinal tract and the remaining bile is
discarded. This enables collection of bile samples for
determination of drug concentration and measurement of
bile flow. It was previously found that TAC was mainly
metabolized by the CYP 3A system in the liver and ex-
creted in bile, whereas only a minor portion of un-
changed TAC was detected in the bile [7,20]. Therefore,
we hypothesized that measuring bile flow together with
TAC and TAC metabolite levels in bile after liver trans-
plantation with biliary drainage would be useful for the
adjustment of an individual patient’s dose and might be a
possible strategy to improve efficacy and reduce toxicity.
To investigate this hypothesis, we determined bile flow
as well as TAC and TAC metabolite levels in bile from
biliary drainage in the early stage after liver transplanta-
tion. This study focused on the relationship among blood
TAC concentration, bile flow, and TAC metabolites in
bile. In addition, the relationship between total bilirubin
(T-Bil), an index of cholestasis, and blood TAC concen-
tration was investigated in liver transplant recipients.
Although several studies have performed retrospective
analysis of patients who have undergone a living-related
liver transplantation [5,21], to our knowledge, this is the
first report describing TAC and TAC metabolite levels in
bile after liver transplantation with biliary drainage.
2. Methods
2.1. Materials
TAC and CyA, which was used as an internal standard in
the liquid chromatography–tandem mass spectrometry
(LC-MS/MS) analysis as described below, were pur-
chased from Sigma-Aldrich Co. (Steinheim, Germany).
We obtained propylene glycol from Wako Pure Chemical
Industries (Osaka, Japan). All other reagents were of
analytical grade and used without further purification.
2.2. Patients
This clinical retrospective study was conducted at the
Department of Transplantation and Regenerative Surgery,
Kyoto Prefectural University of Medicine, Kyoto, Japan.
The study protocol was approved by the Ethics Commit-
tee of the university, and informed consent was obtained
from all patients or their legal guardians (for patients
aged <18 years). Data from the medical records of 16
male and 19 female patients, with a mean age of 38 years
(range, 12 - 59 years), who had received a living-related
liver transplantation at the university from 2004 through
2008 were included.
2.3. Treatment and Study Design
The following immunosuppressive regimen, developed
by the Department of Transplantation and Regenerative
Surgery for use after living-related liver transplantation,
was used in the patients. Initial administration of TAC
(0.3 mg·kg1·day1) was started orally 2 days before
transplantation. On the day of transplantation, TAC (0.1
mg·kg1·day1) was administered by continuous i.v. infu-
sion; this was followed by oral tacrolimus (0.2 mg·kg1
·day1 b.i.d.). During the early stage after surgery (about
1 week), the dose of TAC was adjusted empirically on
the basis of the results of frequent blood level monitoring;
TAC dose was based on the trough blood level (TLTAC)
where target TLTAC was 15 ng·mL1 until 3 weeks after
surgery and subsequently 10 ng·mL1. TAC blood con-
centration was measured with an antibody-conjugated
magnetic immunoassay (ACMIA) method by using a
Dimension Xpand® system. The lower limit of detection
in this method was 1.2 ng·mL 1. Inter- and intra-assay
Copyright © 2013 SciRes. IJCM
Effects of Cholestasis on Whole Blood Concentration of Tacrolimus, an Immunosuppressant,
in Living-Related Liver Transplant Recipients
434
reproducibility were between 5 and 10 (C.V. %) accord-
ing to results of the International Proficiency Testing
Scheme. In 12 patients (6 male, 6 female) in whom bile
samples could be collected, unchanged TAC and de-
methylated and hydroxyated metabolite concentrations in
bile were measured. Bile was collected from biliary
drainage twice a day after transplantation, and volume of
bile was measured gravimetrically to obtain bile flow
rate. The concentrations of TAC and TAC metabolites in
bile were measured by an LC-MS/MS method as de-
scribed below.
2.4. Assay Procedure
Analysis of TAC, demethyl-TAC, and hydroxy-TAC in
bile was performed by LC-MS/MS [22]. The LC-MS/MS
system (Applied Biosystems, Foster City, California)
consisted of an LC-10AD micropump (Shimadzu Cor-
poration, Kyoto, Japan) and an AS8020 automatic sam-
ple injector (Toso, Tokyo, Japan). Mass spectrometry
was performed in the positive-ion electrospray ionization
(ESI) mode. CyA was used as the internal standard.
LC-MS/MS analyses were performed using a Quicksorb
ODS (2.1 mm × 150 mm, 5 μm; Chemco Scientific Co.
Ltd., Osaka, Japan) maintained at 60˚C for all separations.
The mobile phase consisted of 10 mM ammonium ace-
tate/acetonitrile (10:90 v/v) at a flow rate of 0.2 mL/min.
The ion spray voltage and temperature were set at 5000
V and 300˚C, respectively. The flow rates of the nebu-
lizer gas, curtain gas, and collision gas were set at 15, 12,
and 2.0 L·mL1, respectively. Multiple reaction monitor-
ing analysis was performed with transition m/z 821.5
769.7 for TAC and m/z 1202.8425.2 for CyA. Two
metabolites of TAC, demethyl-TAC and hydroxy-TAC,
in bile were measured with transition m/z 807.5755.7
for demethyl-TAC and m/z 837.5785.7 for hydroxy-
TAC. Standard samples were prepared by adding ali-
quots of TAC stock solutions to a drug-free matrix. The
solid-phase extraction cartridges (Oasis® HLB; Waters
Corp., MA, USA) were activated by 1 mL of acetonitrile
followed by 1 mL of ion-exchanged water to prepare the
sorbent for use. Standard and unknown samples (100 μL)
were added to 10 μL of the internal standard (CyA, 200
μg/mL in methanol) and 100 μL of 2% ZnSO4 in 50%
methanol solution, and the tube was vortexed vigorously
for 15 s. After centrifugation for 5 min at 12,000 × g, the
supernatant was applied to the solid-phase extraction
cartridges and subsequently washed with 2 mL of ion-
exchanged water. A 1.5 mL clean tube was positioned
below the solid-phase extraction cartridges, and the
compounds were eluted with 1 mL of acetonitrile. The
eluate was dried with nitrogen at 37˚C. The residue was
redissolved with 100 µL of the mobile phase, and 50 µL
was injected into the LC-MS/MS system for analysis.
2.5. Statistical Analysis
The TAC concentration obtained after 3 days with the
same dosage in each patient was considered the TLTAC
and normalized according to dose (TLTAC/dose). The
ratio of TAC metabolite to TAC in bile (M/P) was de-
termined by the peak area ratio obtained in LC-MS/MS
analysis. Correlations between post operation days (POD)
and M/P, between POD and TLTAC/dose, between
TLTAC/dose and M/P, between bile flow and T-Bil, and
between T-Bile and TLTAC/dose were assessed statistic-
cally using Pearson’s correlation coefficient test. The
differences between the means were considered statistic-
cally significant when p < 0.05.
3. Results
3.1. Relationship among M/P, POD, and
TLTAC/Dose
Figure 1 shows the relationships between M/P and POD,
between M/P and TLTAC/dose, and between TLTAC/dose
and POD in living-related liver transplant patients. There
was no correlation between M/P of demethyl-TAC and
POD, whereas a weak linear correlation was demon-
strated between M/P of hydroxy-TAC and POD (r =
0.345). Moreover, linear correlations were not observed
between M/P and TLTAC/dose, and between TLTAC/dose
and POD.
3.2. Relationship among Bile Flow, T-Bil, and
TLTAC/Dose
Figure 2 shows the relationships between bile flow and
T-Bil, and between TLTAC/dose and T-Bil, in living-re-
lated liver transplant patients. A negative linear correla-
tion was demonstrated between bile flow and T-Bil in
blood (r = 0.495). Furthermore, a positive linear corre-
lation was observed between TLTAC/dose and T-Bil in
blood (r = 0.598). There was no correlation between bile
flow and TLTAC/dose.
4. Discussion
The present study aimed to investigate the effects of
cholestasis on blood TAC levels in liver transplantation
patients. Whereas TAC level was not correlated with bile
flow in our patients with biliary drainage, a positive cor-
relation with T-Bil suggests that T-Bil may be a useful
biomarker for determining the degree of bile duct stric-
ture and TAC dose in liver transplantation patients.
In this study, demethyl- and hydroxy-TAC were
measured in bile because the main metabolites of TAC in
liver-grafted patients are demethyl-TAC in blood and
urine and hydroxy-TAC in bile [23]. It has been reported
that the clearance of TAC is related to postoperative days
Copyright © 2013 SciRes. IJCM
Effects of Cholestasis on Whole Blood Concentration of Tacrolimus, an Immunosuppressant,
in Living-Related Liver Transplant Recipients
Copyright © 2013 SciRes. IJCM
435
demethyl-tacrolimus/tacrolimus, r = -0.063, p = 0.658
hydroxy-tacrolimus/tacrolimus, r = -0.345, p = 0.012
post operation days (day)
0 5 10 15 20 25 30 35
0
1
2
3
4
5
6
7
8
9
10
Metabolite/Parent ratio in bile
(a)
demethyl-tacrolimus/tacrolimus, r = 0.029, p = 0.836
hydroxy-tacrolimus/tacrolimus, r = 0.016, p = 0.910
TL
TAC
/dose (ng/mL/mg)
0 5 10 15 20 25 30 35
0
1
2
3
4
5
6
7
8
9
10
Metabolite/Parent ratio in bile
(b)
r = -0.024, p = 0.618
post operation days (day)
010 2030 40
0
10
TL
TAC
/dose (ng/mL/mg)
50 60
20
30
40
50
60
(c)
Figure 1. Relationships between the ratio of tacrolimus (TAC) metabolite to TAC (M/P) and post operation days (POD) (n =
52) (a), between M/P and TAC trough level normalized dose (TLTAC/dose) (n = 52) (b), and between TLTAC/dose and POD (n
= 421) (c) in living-related liver transplant patients.
Effects of Cholestasis on Whole Blood Concentration of Tacrolimus, an Immunosuppressant,
in Living-Related Liver Transplant Recipients
436
r = -0.495, p < 0.001
T-Bil (mg/dL)
0 2 4 68
0
100
Bile flow (mL/day)
1412
200
300
400
500
600
10 16
(a)
r = 0.598, p < 0.001
T-Bil (mg/mL)
0 5 10 15 20
0
10
TL
TAC
/dose (ng/mL/mg)
35 30
20
30
40
50
60
25
(b)
Figure 2. Relationships between bile flow and total bilirubin (T-Bil) (n = 309) (a) and between tacrolimus (TAC) trough level
normalized dose (TLTAC/dose) and T-Bil (n = 401) (b) in living-related liver transplant.
in pediatric patients receiving living-donor liver trans-
plantation. Fukudo et al. performed population pharma-
cokinetic analysis with nonlinear mixed-effects modeling
and reported that oral clearance of TAC increased with
time in the immediate postoperative period but did not
change further after postoperative day 21 [21]. Antignac
et al. reported that the clearance of TAC was approxi-
mately zero immediately after surgery and then rapidly
increased as a function of postoperative days to reach a
plateau in adult full-liver transplant patients, which was
modeled as a sigmoid relationship [24]. However, in this
study, from 7 to 31 days after surgery, there was no cor-
relation between M/P of demethyl-TAC and POD,
whereas a weak negative correlation was demonstrated
between M/P of hydroxy-TAC and POD. In addition,
linear correlations were not observed between M/P and
TLTAC/dose, and between TLTAC/dose and POD. These
results suggest that improvement of hepatic function and
increase of TAC clearance after postoperative day 7 do
not significantly contribute to hepatic TAC metabolism,
bile excretion, and TLTAC/dose. It is reported that post-
operative biliary stricture is a significant cause of mor-
bidity and mortality in the early stage after liver trans-
plantation and that cholestasis caused by postoperative
biliary stricture induces intestinal bile deficiency and
liver fibrosis [12,14,17]. These observations suggest that
postoperative biliary stricture from liver transplantation
with/without biliary drainage, rather than improvements
Copyright © 2013 SciRes. IJCM
Effects of Cholestasis on Whole Blood Concentration of Tacrolimus, an Immunosuppressant,
in Living-Related Liver Transplant Recipients
437
of hepatic function with recovery from surgical damage
or regeneration of the grafted liver, contributes to inter-
and intra-patient pharmacokinetic variability of TAC.
To evaluate the contribution of postoperative biliary
stricture to pharmacokinetic variability of TAC, the cor-
relations among bile flow, T-Bil, and TLTAC/dose were
investigated in liver transplantation patients. A negative
linear correlation was demonstrated between bile flow
and T-Bil in blood, and a positive linear correlation was
observed between TLTAC/dose and T-Bil in blood. It is
reported that cholestasis caused by bile duct ligation in-
duces intestinal bile deficiency and accumulation of bil-
iary compounds in the body [14]. Moreover, we previ-
ously reported that intestinal absorption of TAC depended
on the amount of bile in the intestine and that lack of bile
after bile duct ligation might result in decreased TAC
absorption; thus, the volume of bile in the intestine was
an important factor in TAC treatment [25]. In addition,
accumulation of bile acids in the liver tissue in biliary
obstruction causes liver fibrosis [15-17]. These results in-
dicate that postoperative biliary stricture is associated
with inter- and intra-patient variability of TLTAC/dose
level after liver transplantation, and that this can be as-
sessed by the T-Bil level. However, in this study, there
was no correlation between bile flow and TLTAC/dose. A
possible explanation for this might be that half of the bile
excreted from the drain was returned to the intestinal
tract during biliary drainage, which might have strongly
affected the absorption of TAC and TLTAC/dose, resulting
in no significant correlation between bile flow and
TLTAC/dose despite a significant linear correlation be-
tween TLTAC/dose and T-Bil in blood. These observa-
tions suggest that T-Bil in blood might be a predictive
biomarker for determining the degree of bile duct stric-
ture and TAC dose in liver transplantation patients, and
that TDM with T-Bil monitoring would be beneficial and
enable individual adjustment of the dose of TAC.
This study had the following limitations: first, a rela-
tively small sample size with study subjects recruited
from a single institution, and second, the use of biliary
drainage, which might have affected absorption and re-
sultant dose of TAC because of return of bile to the in-
testine. These factors limit generalization of the study
findings. Therefore, to evaluate the net effects of choles-
tasis on whole blood concentration of TAC, further in-
vestigation of a large number of subjects in a multicenter
clinical trial is warranted. It would also be interesting to
analyze the effect of return of bile to the intestinal tract in
biliary drainage on the absorption of TAC and resultant
TAC dose with a suitable statistical method.
In conclusion, the present data have several potentially
important implications. First, improvement of hepatic
function and increase of TAC clearance after postopera-
tive day 7 do not significantly contribute to hepatic TAC
metabolism, bile excretion, and TLTAC/dose. Second,
postoperative biliary stricture from liver transplantation
with/without biliary drainage causes inter- and intra-pa-
tient variability of TLTAC/dose level after liver transplant-
tation, and this can be assessed by T-Bil. Thus, T-Bil in
blood might be a predictive biomarker for determining
the degree of bile duct stricture and TAC dose in liver
transplantation patients. Along with an appropriate dos-
ing regimen, TDM including T-Bil would be beneficial
and enable individual adjustment of the dose of TAC in
liver transplantation patients.
5. Acknowledgements
This research received no specific grant from any fund-
ing agency in the public, commercial, or not-for-profit
sectors.
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Abbreviations
ACMIA: Antibody-conjugated magnetic immunoassay;
CyA: Cyclosporine;
CYP: Cytochrome P450;
ESI: Electrospray ionization;
LC-MS/MS: The liquid chromatography-tandem mass
spectrometry;
M/P: The ratio of TAC metabolite to TAC in bile;
Pgp: P-glycoprotein;
POD: Post operation days;
TAC: Tacrolimus;
T-Bil: Total bilirubin;
TDM: Therapeutic drug monitoring;
TLTAC: Trough blood level;
TLTAC/dose: TAC trough level normalized dose.
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