Simultaneous Determination of Bilirubin and Its Glucuronides in Liver

Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159

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Journal of Pharmaceutical and Biomedical Analysis

j ournal homepage: www.elsevier.com/locate/jpba

Simultaneous determination of bilirubin and its glucuronides in liver

microsomes and recombinant UGT1A1 enzyme incubation systems by

HPLC method and its application to bilirubin glucuronidation studies

∗,1 1 ∗∗

Guo Ma , Jiayuan Lin , Weimin Cai, Bo Tan, Xiaoqiang Xiang, Ying Zhang, Peng Zhang

Department of Clinical Pharmacy, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Bilirubin, an important endogenous substances and liver function index in humans, is primarily

Received 8 August 2013

eliminated via UGT1A1-catalyzed glucuronidation. Instability of bilirubin and its glucuronides brings

Received in revised form 17 January 2014

substantial technical challenges to conduct in vitro bilirubin glucuronidation assay. In the present study,

Accepted 18 January 2014

we developed a simple and robust HPLC method for simultaneous determination of unconjugated biliru-

Available online 27 January 2014

bin (UCB) and its multiple glucuronides, i.e. bilirubin monoglucuronides (BMGs, including BMG1 and

BMG2 isomers) and diglucuronide (BDG) in rat liver microsomes (RLM), human liver microsomes (HLM)

Keywords:

and recombinant human UGT1A1 enzyme (UGT1A1) incubation systems, and applied it to study in vitro

Bilirubin

Glucuronides bilirubin glucuronidation. UCB, BMG1, BMG2, BDG and their isomers in the incubation mixtures were

UGT1A1 successfully separated using a C18 column with UV detection at 450 nm and mobile phase consisted

Liver microsomes of 0.1% formic acid in water and acetonitrile by a linear gradient elution program. Assay linearities of

HPLC bilirubin were conﬁrmed in the range 0.01–2 ␮M. Precision of UCB, BMG1, BMG2 and BDG (n = 5) at low,

medium and high concentration was within the range of RSD 0.4–3.7%, accuracy expressed in the mean

assay recoveries of them (n = 5) ranged from 92.8 ± 1.5% to 104.3 ± 2.2% for intra- and inter-day assays

and the mean extraction recoveries of them (n = 5) were above 91.5 ± 1.0%. Stability of bilirubin and its

◦ ◦

glucuronides was satisfactory at 37 C in the incubation solutions during the reaction (30 min), 25 C for

◦

24 h and −70 C for 7 d in the processed incubation samples with methanol. Furthermore, we established

stable, reliable in vitro incubation systems and optimized the incubation conditions to characterize the

kinetics of bilirubin glucuronidation by RLM, HLM and UGT1A1, respectively. The kinetic parameters of

formation of total bilirubin glucuronides (TBG, the sum of BMG1, BMG2 and BDG) were as follows: Km of

0.45 0.016, 0.40 ± 0.022, 0.44 ± 0.018 ␮M, Vmax of 2.65 ± 0.057, 1.86 ± 0.029, 2.95 ± 0.036 nmol/mg/min,

CLint of 5.92 ± 0.22, 4.70 ± 0.079, 6.72 ± 0.27 mL/mg/min by RLM, HLM and UGT1A1, respectively. Biliru-

bin glucuronidation obeyed the Hill equation by RLM and the Michaelis–Menten equation by HLM and

UGT1A1 in the range of substrate concentration selected, respectively. In addition, the relative propor-

tions between BDG and BMGs were in connection with enzyme sources (e.g. RLM, HLM and UGT1A1) and

bilirubin concentration.

Abbreviations: UCB, unconjugated bilirubin; CB, conjugated bilirubin; BG, bilirubin glucuronides; BMGs, bilirubin monoglucuronides; BDG, bilirubin diglucuronide; TBG,

total bilirubin glucuronides; UGT(s), UDP-glucuronosyltransferase(s); UGT1A1, UDP-glucuronosyltransferases1A1; RLM, rat liver microsomes; HLM, human liver microsomes;

UDPGA, uridine diphosphoglucuronic acid; DMSO, dimethylsulfoxide; MRP2, multidrug resistance-associated protein 2; HPLC, high performance liquid chromatogra-

phy; QC, quality control; LOD, limit of detection; LLOQ, lower limit of quantiﬁcation; RSD, relative standard deviation; Conc., concentration(s); V, reaction velocity; Km,

Michaelis–Menten constant; Vmax, maximum reaction velocity; CLint, intrinsic clearances; R , residual sum of squares; AIC, Akaike information criterion; CDER, Center for

Drug Evaluation and Research.

∗

Corresponding author. Tel.: +86 21 51980025; fax: +86 21 51980001.

∗∗

Corresponding author. Tel.: +86 21 51980024; fax: +86 21 51980001.

E-mail addresses: [email protected], [email protected] (G. Ma), [email protected] (P. Zhang). 1

Co-ﬁrst authors.

http://dx.doi.org/10.1016/j.jpba.2014.01.025

150 G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159

1. Introduction kernicterus, Crigler–Najjar syndromes (Types I and II), Gilbert’s syn-

drome, and even death [1,19–21]. As a result, in drug discovery,

Bilirubin is the principal constituent of mammalian bile pigment development and use settings, the in vitro ability of drug to inhibit

and end-product of heme catabolism. Approximately 250–300 mg bilirubin glucuronidation is commonly evaluated. In addition,

of bilirubin is produced in a normal adult each day. Bilirubin is some xenobiotics (e.g. phenobarbital, dexamethasone, rifampicin

an important index of liver function and biomarker of hepato- and herbal extracts Yin zhi huang) can also induce UGT1A1

toxicity, as well as an important clinical basis for determining gene expression and enhance UGT1A1 activity by a number of

jaundice. As an essential endogenous substance in humans and multifunctional nuclear receptors such as constitutive androstane

animals, bilirubin was long thought to be a non-functional and receptor (CAR), pregnane X receptor (PXR), glucocorticoid recep-

toxic waste product. Recent studies [1–3] have shown that biliru- tor (GR), aryl hydrocarbon receptor (AhR), and hepatocyte nuclear

bin has multiple biological functions in animals and plants, for receptor 1␣ (HNF1␣) [22–25]. These factors contribute to pro-

example, potent antioxidant and cytoprotective effects at physi- mote bilirubin glucuronidation, and reduce serum UCB level. They

ological and mildly elevated concentrations, as well as activation might have important clinical application in preventing and treat-

of heme oxygenase, and can protect against cardiovascular dis- ing unconjugated hyperbilirubinemia and neonatal jaundice.

eases (e.g. atherosclerosis) and tumor development. However, it It is not difﬁcult to see that establishing a simple and robust

can cause apoptosis, cytotoxicity and neurotoxicity at markedly assay method for accurate measurement of bilirubin and its

elevated plasma and tissue bilirubin levels, and result in severe, glucuronides is vitally important for us to study bilirubin glu-

irreversible brain and neurological damage (e.g. kernicterus), espe- curonidation and its inhibition or induction, which has important

cially in neonates [1,4–7]. clinical signiﬁcance in diagnosis, prevention and treatment of

Bilirubin is mainly metabolized by liver. Before it is transported bilirubin-related malady or toxic reaction, for example, jaundice,

into liver, bilirubin exists mostly in the form of unconjugated biliru- hyperbilirubinemia and kernicterus. However, as a weakly polar,

bin (UCB) and binds highly to albumin in the blood. After hepatic poorly soluble compound, bilirubin is very labile. It is highly photo-

uptake, UCB is extensively metabolized to bilirubin glucuronides sensitive and readily oxidized, rapidly degraded in both acidic and

(BG) by UDP-glucuronosyltransferases1A1 (UGT1A1) localized pri- alkaline solutions, and high-afﬁnity for proteins (e.g. serum albu-

marily in smooth endoplasmic reticulum of hepatocyte. In this min), as well as strong adsorption on experimental equipments and

glucuronidation reaction, a glucuronosyl moiety is conjugated to materials (e.g. nonspeciﬁc binding of bilirubin to walls of the plastic

one of the propionic acid side chains, located on the C8 and pipes, tips, vials and tubes, as well as the chromatographic channel

C12 carbons of the two central pyrrole rings of bilirubin, result- and column) [19,26,27]. Equally as problematic is the instability of

ing in producing two bilirubin monoglucuronides (BMGs) isomers bilirubin glucuronides, especially BMGs. In aqueous media, BMGs

(i.e. BMG1 and BMG2). BMGs were further glucuronidated, and was rapidly transformed into BDG and UCB by dipyrrole exchange

formed bilirubin 8,12-diglucuronide (BDG) [8] (Fig. 1). In adult mechanism [28]. Furthermore, bilirubin itself is composed of three

humans, over 80% of the bilirubin conjugates are normally BDG isomers (i.e. bilirubin IX-␣, XIII-␣ and III-␣), and bilirubin glu-

[9], whereas BMGs predominate in newborns [10]. Finally, BG curonidation involves a sequential reaction that produces multiple

(i.e. BMGs and BDG) formed are secreted into bile by multidrug glucuronides (i.e. BMG1, BMG2, BDG and their isomers), resulting

resistance-associated protein 2 (MRP2), and subsequently elimi- in difﬁcult quantitation of glucuronidation assay and establishment

nated via feces and urine [11]. of initial rate condition, if not given particular attention. All these

UGT1A1 is a critical enzyme responsible for metabolism and factors, especially, in vitro instability, bring the substantial technical

detoxiﬁcation of bilurubin [12]. Glucuronidation by UGT1A1 is challenges in bilirubin glucuronidation [28,29]. These challenges

an essential step for bilirubin elimination [13]. Xenobiotics (e.g. have been manifested in signiﬁcant disparities in estimated kinetic

SN-38 [14], atazanavir, indinavir [15,16], erlotinib [17], sorafenib parameters and mechanism for bilirubin glucuronidation. Three

[18]) inhibiting UGT1A1, and genetic variants resulting in partial groups [26,30,31] reported that bilirubin glucuronidation obeyed

or complete loss of UGT1A1 activity, can cause disorder of biliru- Michaelis–Menten kinetics. One group [32] reported it exhibited

bin metabolism, and lead to accumulation of bilirubin in blood substrate inhibition kinetics, and the other group [33] reported it

and/or brain, which further result in jaundice, hyperbilirubinemia, obeyed Michaelis–Menten kinetics at low protein concentration

H2C CH3 OHHO

H3C N N CH2

UGT1A1 N HN UGT1A1 H2C CH3 H3C CH3 OHHO H2C HOOC CO2H CH3 H3C OHHO O OH N N CH2 O H3C OH O HO N N CH2 N HN

H3C CH3 N HN BMGs HO2C CO2H H3C CH3 HO O O OH H2C HOOC COOH CH3 HO O O OH OHHO OH O O HO UCB H3C BDG N N CH2 UGT1A1 UGT1A1 N HN

H3C CH3 HO2C COOH HO O HO O

OH O

Fig. 1. The molecular structures of bilirubin and its glucuronides. UCB was metabolized to BMGs (including two isomers BMG1 and BMG2), and BMGs was further metabolized

to BDG by UGT1A1. UCB, unconjugated bilirubin; BMGs, bilirubin monoglucuronides; BDG, bilirubin diglucuronide; UGT1A1, recombinant human UGT1A1 enzyme.

G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159 151

of 0.05 mg/mL and Hill equation at high protein concentration 2.3. Preparation of rat liver microsomes

of 0.5 mg/mL. Likewise, estimates of Km and Vmax ranged from

0.20–24 ␮M and 0.08–1.08 nmol/min/mg, respectively. These dif- Six Sprague-Dawley rats (male and female, 200 ± 20 g) were

ferences may be caused by different incubation condition selected provided by SIPPR-BK Laboratory Animal Co., Ltd. (Shanghai, China,

(e.g. source and concentration of enzyme, concentration range of Animal study protocol number: 2008001626534). All animal stud-

substrate, reaction time), and some inﬂuential factors (e.g. light, ies were performed according to the requirement of the National

heat, oxygen, pH, protein binding, physical adsorption and other Act on the Use of Experimental Animal (China) that was approved

factors) in assaying of bilirubin and its glucuronides. by the Ethics Committee for Animal Experiment of School of Phar-

In order to carefully characterize the kinetics of bilirubin glu- macy, Fudan University in Shanghai.

curonidation, we develop a speciﬁc, sensitive and robust HPLC RLM were prepared as previously reported [40] with slight mod-

method for simultaneous determination of bilirubin and its glu- iﬁcations. The SD rats were fasted for 12 h before the animal test

curonides in rat liver microsomes (RLM), human liver microsomes was conducted. They were sacriﬁced by decapitation, and were

(HLM) and recombinant human UGT1A1 enzyme (UGT1A1) incuba- rapidly perfused with ice-cold 0.2 mol/L PBS (pH 7.4) via the por-

tion systems, and established and optimized the in vitro incubation tal vein to ﬂush the liver. The livers were removed and placed in

conditions in the present study, respectively. Especially, compared the ice-cold PBS at once, washed away the redundant blood and

with the previous methods [28,29,33–39], we simultaneously blotted the moisture. After that, the tissue was minced into the

determined bilirubin, bilirubin glucuronides and their multiple iso- slices. The slices were weighed and added the PBS (four times

mers in three incubation matrix, and disclosed the differences of of the weight of the tissue), homogenated with GF-1 dispersator

kinetic mechanism of bilirubin glucuronidation by RLM, HLM and (Kylin-Bell, Haimen, Jiangsu, China). The homogenate was cen-

UGT1A1. The in vitro study will provide an important reference for trifuged in a MICROCL 17R centrifuge (Thermo scientiﬁc, Boston,

◦

in vivo bilirubin metabolism, and has the potential application in MA, USA) at 9000 × g for 20 min at 4 C and the supernatant was

diagnosis, prevention and treatment of bilirubin-related malady or ultracentrifuged employing a CP-WX ultracentrifuge (TECHCOMP

◦

toxic reaction. Ltd., Shanghai, China) at 100,000 × g for 1 h at 4 C in order to obtain

the microsomal pellet. The obtained pellet was re-suspended in 30%

◦

−

2. Materials and methods glycerol–0.2 mol/L PBS (pH 7.4) and stored at 70 C until use. The

protein concentration was determined by a BCA kit (Cwbiotech,

2.1. Chemicals and reagents

Shanghai, China).

Bilirubin (including three mixed isomers, i.e. bilirubin

2.4. Preparation of samples

␣

IX- 90.11%, XIII-␣ 3.12% and III-␣ 5.93%), uridine 5 -

diphosphoglucuronic acid trisodium salt (UDPGA) and alamethicin 2.4.1. Bilirubin stock solution

were purchased from Sigma–Aldrich (China-mainland). Ascorbic Bilirubin stock solution was prepared by dissolving bilirubin in

acid was provided by Aladdin Chemistry Co., Ltd. (Shanghai, China). 100% dimethyl sulfoxide (DMSO) to yield concentration of 2 mM,

◦

−

Formic acid, MgCl2·6H2O, K2HPO4·3H2O, KH2PO4, NaH2PO4·2H2O, then rapidly aliquoted and stored at 70 C.

Na2HPO4·12H2O, NaCl (all of analytical grade) were purchased

from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.4.2. Standard and quality control (QC) samples

Acetonitrile and methanol (both of HPLC grade) were provided The standard and QC samples (n = 5) were prepared as described

by Fisher Scientiﬁc International, Inc. (Fair Lawn, NJ, USA). Argon in Section 2.6. As the incubation mixtures, they contained

was purchased from Shanghai Lvmin Gas Co. Ltd. Puriﬁed water bilirubin (ﬁnal concentration of 0.05–2 ␮M, dissolved in 100%

was prepared in a water puriﬁcation system (EMD Millipore Corp., DMSO), RLM (HLM or UGT1A1, ﬁnal concentration of 12.5 ␮g

Billerica, MA, USA). All other reagents were of analytical grade at of protein/mL), potassium phosphate buffer (50 mM, pH 7.4),

least. MgCl2·6H2O (0.88 mM), alamethicin (22 ␮g/mL), as well as in the

Pooled SD rat liver microsomes (RLM) were prepared in our lab- absence or presence of UDPGA (3.5 mM). The standard samples

oratory. Pooled human liver microsomes (HLM) were purchased were the simulated incubation mixtures in the absence of UDPGA.

from CELSIS, Inc. (Chicago, IL, USA). Recombinant Human UGT1A1 The QC samples representing the initial low, medium and high con-

enzyme (UGT1A1, BD-Supersomes ) was purchased from BD centrations of bilirubin were set at 0.05, 0.2, 1.5 ␮M for assessing

Biosciences-Discovery Labware (Woburn, MA, USA). the precision, accuracy, recovery and stability of bilirubin standard

solution, and 0.2, 0.75, 1.5 ␮M for assessing the stability of biliru-

2.2. Chromatographic conditions bin and its glucurconides in the incubation solutions during the

reaction and the processed incubation samples (the latter are

Chromatographic analyses were performed on a Shimadzu LC- deproteinized and extracted by addition of methanol containing

2010A HT HPLC system (Kyoto, Japan) equipped with a quaternary ascorbic acid). Because of lack of commercial products of BMG1,

pump, an automatic sampler, a UV–vis detector, a system controller BMG2 and BDG, the samples for stability of glucoronides have to

and a temperature control oven. System control and data anal- be prepared by bilirubin glucuronidation reaction as described in

yses were carried out using a Shimadzu LC solution workstation Section 2.6 with slight modiﬁcations. Namely, the reaction was ter-

(Shimadzu, Kyoto, Japan). minated by the addition of ice-cold methanol (a quarter volume of

Bilirubin and its glucuronides were separated on a HPLC col- the reaction mixture, in the absence of ascorbic acid). The incuba-

umn (reverse phase Diamonsil C18 column, 200 mm 4.6 mm, tion mixtures were immediately freeze-dried using Liquid Nitrogen

␮

i.d., 5 m particle size, Dikma) with guard column (Cartridge Guard Vacuum Freeze Dryer (Tofﬂon, Shanghai, China) which need not to

Column E, Inertsil ODS-SP, 10 mm 4 mm, GL Sciences Inc.). The been deproteinized. The freeze-dried powders were re-dissolved

mobile phase consisted of 0.1% formic acid in water (A) and 100% with water, then were used to evaluate the stability of bilirubin

◦

acetonitrile (B) was delivered at a ﬂow rate of 1 mL/min. The lin- and its glucurconides at 37 C. Finally, the samples were processed

ear gradient elution program was as follows: 0–9 min, 40–75% as described in Section 2.4.3. The processed incubation samples

◦ ◦

B; 9–18 min, 75–95% B; 18–27 min, 95% B; 27–30 min, 95–40% B. with methanol for stability at 25 C and −70 C were prepared as

◦

The column temperature was 45 C. The detection wavelength was described in Section 2.6. All these samples were prepared in amber

␮

450 nm. The sample injection volume was 100 L. glass vials with screw cap, and processed in a dim light room.

152 G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159

◦

2.4.3. Extraction procedure and −70 C (frozen temperature) for 0, 10, 20, and 30 d (long-

␮

A 600 L ice-cold methanol containing 200 mM ascorbic acid term stability), respectively. The bilirubin standard solutions (in

was added to the above standard or QC samples (200 ␮L), respec- the absence of UDPGA) at the concentration level of 0.05, 0.2 and

␮

tively. The mixtures were vortexed for 2 min and then centrifuged 1.5 M were prepared as described in Section 2.5.2 and stored at

◦

at 12,000 rpm for 10 min to precipitate and separate protein. The 25 C for 0, 6, 12, and 24 h. The stability of UCB, BMG1, BMG2 and

◦

supernatant was injected into the HPLC system for analysis. BDG was evaluated at 37 C (incubation temperature, assessing the

short-term stability of bilirubin and its glucurconides in the incu-

2.5. HPLC validation procedures bation solutions during the reaction) for 0, 5,10, 15, 30, and 60 min,

◦

25 C (assessing the post-preparative stability of the processed

◦

−

2.5.1. Selectivity samples with methanol) for 0, 4, 8, 12, 24 h, and 70 C (assessing

Selectivity of the method was evaluated by analyzing the blank the long-term stability of the processed samples with methanol)

samples (the incubation samples in the absence of bilirubin, i.e. for 0, 3, 7 d from the RLM, HLM and UGT1A1 incubation samples

the incubation matrix described in Section 2.4.2), standard samples (n = 3), respectively. The samples for bilirubin glucuronidation dur-

(the incubation samples in the absence of UDPGA) and bilirubin ing and after the reaction were prepared as described in Section

glucuronidation samples (the incubation samples in the presence of 2.4.2. The ﬁnal concentrations of bilirubin in these incubation mix-

␮

bilirubin and UDPGA) from the RLM, HLM and UGT1A1 incubation tures were 0.2, 0.75 and 1.5 M at the initial reaction time point.

systems, respectively. The samples were prepared as described in All these samples were processed as described in Section 2.4.3. The

Section 2.4. peak areas of UCB, BMG1, BMG2 and BDG for stability of the test

samples at different time points were compared with the peak area

2.5.2. Calibration curves and linearity of that at 0 min. All the stability determinations use a set of samples

The standard samples for calibration curves (n = 3) were pre- prepared from freshly made stock solution of bilirubin.

pared as described in Section 2.4. The ﬁnal concentrations of

bilirubin in the standard samples were 0.01, 0.05, 0.1, 0.2, 0.5, 1, 1.5

2.6. Bilirubin glucuronidation

and 2 ␮M, respectively. The combined peak areas of bilirubin (i.e.

the sum of peak areas of bilirubin IX␣, XIII␣ and III␣ isomers) were

The incubation procedure for bilirubin glucuronidation were as

plotted against the standard concentrations to establish the calibra-

follows: (1) bilirubin (ﬁnal concentration range of 0.25–10 ␮M),

tion curves. Quantitation of BMG1, BMG2 and BDG was based on

potassium phosphate buffer (50 mM, pH 7.4), MgCl2·6H2O

the standard curve of bilirubin as the previous reports [32,33]. Like-

(0.88 mM), alamethicin (22 ␮g/mL) and RLM (HLM or UGT1A1, ﬁnal

wise, peak areas of these glucuronides for quantitation were also

concentration 12.5–50 ␮g of protein/mL) were mixed in amber

◦

the sum of peak areas of their isomers in the study, respectively.

glass vials (full of argon) and pre-incubated at 37 C for 2 min in a

Limit of detection (LOD) was calculated as the ﬁnal concentration

shaking water bath; (2) the reaction was initiated by the addition of

of bilirubin producing a signal-to-noise ratio of 3. The lower limit of

UDPGA (3.5 mM); (3) the mixture (total volume 200 ␮L) was incu-

◦

quantiﬁcation (LLOQ) was considered as the lowest concentration

bated at 37 C for 0–60 min; and (4) the reaction was terminated by

of the calibration curve.

the addition of 600 ␮L ice-cold methanol containing 200 mM ascor-

bic acid. Bilirubin and its glucuronides in the incubation mixture

2.5.3. Precision, accuracy and recovery

were extracted as described in Section 2.4.3. Bilirubin was dissolved

Precision and accuracy of the analytical method were evaluated

in DMSO just before adding into the incubation mixtures. The ﬁnal

by analyzing QC samples at three initial concentration levels (0.05,

DMSO concentration in the incubation mixture was 1%. The sam-

0.2 and 1.5 ␮M bilirubin). Precision was expressed using relative

ples were stored in amber glass vials, handled and processed under

standard deviation (%, RSD), and accuracy was deﬁned as per-

the dim light. All experiments were performed in triplicates.

cent of deviation between the true and the measured value, which

both required to be measured using ﬁve determinations (n = 5) per

2.7. Kinetic analysis

concentration. To assess intra-day precision and accuracy, the QC

samples were measured within one day. For inter-day assays, QC

Kinetic analysis was performed by ﬁtting the Michaelis–Menten

samples were analyzed for three consecutive days. The precision

equation (Eq. (1)) or the Hill equation (Eq. (2)) to the kinetic

was required within 15% of the RSD, and accuracies were required

data (substrate concentrations and initial rates) with SigmaPlot

not to exceed ±15% of the actual value at three concentration lev-

12.0 (Systat Software Inc., San Jose, CA). Glucuronidation velocity

els [41]. In addition, within-run and inter-run precision for BMG1,

(V) in Eqs. (1) and (2) was calculated as nanomoles of glu-

BMG2 and BDG from the same batch of RLM, HLM and UGT1A1 incu-

curonide(s) formed per mg protein amount per reaction time

bation samples at three initial bilirubin concentration levels of 0.2,

(nmol/mg protein/min). The kinetic parameters Vmax and Km (also

0.75 and 1.5 ␮M was only assessed owing to in the absence of com-

depicted as S50 in Eq. (2)) are deﬁned as the maximum velocity and

mercially supplied BMG1, BMG2 and BDG as reference substance

the substrate concentration at which velocity equals to half of the

hereon.

Vmax, respectively. n in Eq. (2) is the Hill coefﬁcient, indicative of

Extraction recovery of bilirubin was determined by comparing

the degree of curve sigmoidicity and/or cooperativity. The kinetic

the chromatographic peak areas of the analytes extracted from ﬁve

parameters CLint (intrinsic clearance, =Vmax/Km) was calculated as

replicate QC samples at three concentration levels (0.05, 0.2, 1.5 ␮M

the rate of disappearance of the test compound in units of mL/mg of

bilirubin) to that of the pure bilirubin solutions without extraction

protein/min [42]. Model appropriateness was determined by visual

procedure at the same nominal concentrations. The pure biliru-

inspection of the Eadie–Hofstee plots, comparison of the residual

bin solutions were prepared by dissolving bilirubin in the mixed 2

sum of squares (R ) and Akaike information criterion (AIC) values

solvent DMSO–methanol (1:4, v/v).

[43,44]. V × S 2.5.4. Stability V max [ ]

= (1)

The stability was thoroughly evaluated by analyzing bilirubin Km + [S]

stock solutions, standard solutions and QC samples exposed to dif- n

Vmax × [S]

ferent conditions. The bilirubin stock solution (2 mM) was stored at V = n (2) ◦ Kn

+ [S]

25 C (room temperature) for 0, 2, 4, and 6 h (short-term stability) m

G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159 153

2.8. Statistical analysis 6000 RLM

5000

Statistical analyses were performed by one-way ANONA, two-

way ANONA and T-test using GraphPad Prism V5 software for 4000

UCB

Windows (GraphPad Software, San Diego, CA). Differences were BMG2

3000

considered signiﬁcant when p values were less than 0.05 (p < 0.05). BMG1 BDG

2000 C1

3. Results and discussion 1000 B1 A1

3.1. Analytical methods

0.0 5.0 10.0 15.0 20.0 25.0 min

In the present study, we simultaneously determined biliru- uV

bin and its multiple glucuronides in the RLM, HLM and UGT1A1 6000 HLM

incubation systems by an HPLC method, and carried out the ana-

5000

lytical method validation including selectivity, linearity, sensitivity,

4000 accuracy, precision and stability. Although a number of assay meth- UCB

BMG2

ods for bilirubin and/or its glucuronides in some matrix such as 3000 BMG1

BDG

serum/plasma, bile, or cell have been established for the past

2000

decades [28,29,33–39], different approaches are needed for the C2

determination of bilirubin in different species and matrix. The con- 1000 B2

ventional analytical approaches, for example, diazo assay and direct A2

spectrophotometry were usually employed to determine concen-

trations of free and conjugated bilirubin in biological ﬂuids and 0.0 5.0 10.0 15.0 20.0 25.0 min

tissues (e.g. brain or blood). The former was based on the reac- uV

6000 UGT1A1

tion of diazotized sulfanilic acid with bilirubin to form azobilirubin,

which formed the quantitative basis of bilirubin in biological ﬂuids. 5000

The latter was based on measuring the absorbance of bilirubin near

4000 BMG2

460 nm [45]. Compared with the newly established HPLC method, UCB BMG1

BDG

the two methods exhibited poor selectivity. It was difﬁcult for 3000

them to simultaneously separate and determine UCB, BMG1, BMG2, 2000

BDG and their multiple isomers. Moreover, radioassay was used to

1000 B3

assess bilirubin levels in brain and CSF after intravenous adminis-

14 A3

tration of [ C]-UCB to Gunn rats or guinea pigs [46,47]. ELISA using 0

an anti-bilirubin antibody was also employed to assay bilirubin and

0.0 5.0 10.0 15.0 20.0 25.0 min

its oxidation in CSF of Alzheimer’s disease patients and in the rat

intestinal mucosa [48–50]. It is a pity that the last two methods

Fig. 2. Representative chromatograms for bilirubin glucuronidation in RLM, HLM,

are not generally accessible due to the commercial unavailabil-

UGT1A1 incubation systems, respectively. A1–A3 represent blank samples (the incu-

ity of radiolabeled bilirubin or anti-bilirubin antibody, and, more

bation samples in the absence of bilirubin); B1–B3, standard samples (the incubation

importantly, underestimate UCB concentrations due to incomplete samples in the absence of UDPGA); C1–C3, bilirubin glucuronidation samples (the

incubation samples in the presence of bilirubin and UDPGA) in the RLM, HLM,

extraction of the analytes from tissues and organs. In previous assay

UGT1A1 incubation systems, respectively. RLM, rat liver microsomes; HLM, human

[38], bilirubin glucuronides and total bilirubin in biological ﬂu-

liver microsomes; UGT1A1, recombinant human UGT1A1 enzyme. The peaks at

ids and tissues were usually determined by a complex hydrolysis

24.714, 25.279 and 25.761 min were assigned as bilirubin III-␣, IX-␣ (major peak)

step, i.e. hydrolytic reagents (e.g. NaOH) were added into the sam- and XIII-␣, respectively. The peaks at 8.564 and 8.940 min were assigned as the

␣ ␣

ples consisted of bilirubin glucuronides, and then hydrolyzed these BMG1 IX- (major peak) and XIII- , respectively. The peaks at 7.571 and 7.950 min

were assigned as the BMG2 III-␣ and IX-␣ (major peak), respectively. The peaks at

glucuronides into UCB. The procedure involved extraction, separa-

4.515, 4.914 and 5.346 min were assigned as the BDG III-␣, IX-␣ (major peak) and

tion, hydrolysis and neutralization of samples, easily resulting in

XIII-␣, respectively. Incubations were conducted with 1 ␮M bilirubin at 12.5 ␮g/mL

loss of the analytes and inaccurate quantiﬁcation. Furthermore, the

microsomal (or UGT1A1) protein concentration for 15 min; chromatographic con-

indirect method cannot separate and determine the speciﬁc conditions were described in Section 2.2.

stituents of bilirubin glucuronides (i.e. BMG1, BMG2, BDG and their

isomers). In a word, we simultaneously determined bilirubin and its

multiple glucuronides (including their multiple isomers) in three

identiﬁcation of UCB, BMG1, BMG2, BDG and their isomers were

different incubation systems by a simple, reliable and reproducible

based on their lipophilicity and polarity, as well as the elution pat-

HPLC method, and ﬁrst applied it to systematically investigate and

tern, chromatographic peak position, relative retention time from

disclose the differences of kinetics of bilirubin glucuronidation by

previous reports [26,28,32,33] and our current study. As shown in

RLM, HLM and UGT1A1 in the present study. In addition, compared

Fig. 2, UCB (including UCB III-␣, IX-␣ and XIII-␣) was metabolized

with the previous assay [28,29,33–39], the processed procedure of

to BMG1 (including BMG1 IX-␣ and XIII-␣), BMG2 (including BMG2

samples (e.g. preparation and extraction) was simple, rapid and ␣

III- and IX-␣) and BDG (including BDG III-␣, IX-␣ and XIII-␣).

reproducible.

All these analytes including their multiple isomers were efﬁciently

separated on the HPLC column. No interference was observed at the

retention times of each analyte in any incubation samples used for

3.2. Selectivity

analysis. Although the monoglucuronides BMG1 and BMG2 were

efﬁciently separated, it was difﬁcult to use the techniques described

The representative chromatograms for bilirubin glucuronida-

here to assign with certainty the propionic acid side-chain (C or

tion by RLM, HLM and UGT1A1 are similar (Fig. 2). Ten peaks 8

C ) position for the BMGs peaks. In a word, the HPLC method

from bilirubin and its glucuronides including their isomers 12

exhibited good selectivity and high resolution.

were detected in the incubation samples. Peak assignment and

154 G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159

Table 1

Regression equations, LOD, LLOQ and linear range for bilirubin.

Incubation systems Regression equations r LOD (␮M) LLOQ (␮M) Linear range (␮M)

RLM Y = 72186X − 589.32 0.9996 0.005 0.01 0.01–2

HLM Y = 70403X − 276.16 0.9998 0.005 0.01 0.01–2

UGT1A1 Y = 70182X + 283.75 0.9998 0.005 0.01 0.01–2

3.3. Calibration curve and linearity remaining percentages of bilirubin at the concentration of 0.05, 0.2

and 1.5 ␮M were ≥96.9 ± 5.5% (Table S2 in the supplementary data).

The regression equations for calibration curves, LOD, LLOQ and Stability of bilirubin and its glucuronides (i.e. UCB, BMG1, BMG2

◦

linear range for bilirubin were shown in Table 1, respectively. The and BDG) was satisfactory at 37 C during the reaction (0–30 min) in

◦

HPLC method showed good linearities in the range of 0.01–2 ␮M the RLM, HLM and UGT1A1 incubation solutions, 25 C for 24 h and

− ◦

bilirubin with correlation coefﬁcients (r) ≥ 0.9996 for three cali- 70 C for 7 d in the processed incubation samples with methanol

bration curves. The linearities became poor when concentration of (Table 3 and Tables S3–S11 in the supplementary data). These stud-

bilirubin was beyond 2 ␮M. Hereon, it was possibly due to strong ies indicated that bilirubin and its glucuronides were stable in the

adsorption and overload (or saturation) of bilirubin on the chro- procedure of preparation, incubation, handling, storage and assay

matographic column. The LLOQ for bilirubin was set at the lowest of the tested samples under the conditions selected. The satisfac-

concentration in the linear standard curve and equal to 0.01 ␮M, tory stability was probably due to a series of measures that we

and the accuracies for bilirubin were 105.9 ± 5.1%, 93.4 ± 3.8% and took. In order to protect from photolysis, oxidation, degradation

92.9 ± 3.6% while the precisions (RSD, %) were 4.8%, 4.0% and 3.9% and adsorption of bilirubin and its glucuronides, we kept the prepa-

in the RLM, HLM and UGT1A1 incubation system, respectively. ration, incubation, extraction and storage of the tested samples in

It must be pointed out that, bilirubin glucuronides (i.e. BMG1, amber glass containers ﬁlled with argon in the dark room equipped

BMG2 and BDG) are extremely unstable and in the absence of com- with dim yellow light (without UV), under the near-neutral (pH 7.4)

mercial products. Because the added glucuronic acid moiety does incubation circumstance, addition of the antioxidant and stabilizer

not absorb light at 450 nm wavelength, the molar extinction coef- (e.g. ascorbic acid) in the incubation mixtures, sample storage at

◦

ﬁcient of the parent compound UCB is not affected. UCB, BMG1, low temperature (−70 C), usage of low adsorptive and/or light-

BMG2 and BDG have the same molar extinction coefﬁcient, and resistant experimental materials (e.g. low-binding tips, aluminum

their calibration curves are extremely similar, therefore the cal- foil-wrapped amber glass vials, tubes with screw cap and ﬂask

ibration curves for UCB were used to estimate concentration of with glass stopper), as well as quick manipulations in the experi-

BMG1, BMG2 and BDG as previous reports [32,33,46,51]. Moreover, ment. Among them, ascorbic acid (as a reducing agent) can strongly

quantiﬁcation of the analytes only based on the UCB standard curve inhibit non-enzymic hydrolysis of bilirubin glucuronides, and com-

simpliﬁed the quantiﬁcation process. pletely diminish the conversion of BMGs into BDG and UCB, as

well as prevent from oxidation of bilirubin and its glucuronides

[28]. Meanwhile, the satisfactory stability of bilirubin and its glu-

3.4. Precision, accuracy and recovery

curonides in the incubation solutions during the reaction was

probably related with the stabilizing effect of the protein from the

The method for determining bilirubin in the RLM, HLM and

RLM (HLM or UGT1A1). The protein can strongly inhibit the con-

UGT1A1 incubation samples was validated according to FDA guide-

version of BMGs into BDG and UCB [33]. It must be pointed out that

lines for the analysis of drugs in biological ﬂuids. The assay results

any decreased time for pretreatment and analysis of samples would

showed that all the intra- and inter-day precision for bilirubin

decrease the possibility of test sample degradation. In addition, the

did not exceed 3.7% of RSD at three concentration levels. Data

HPLC column and channel were eluted using 100% acetonitrile over

on accuracies about the mean assay recoveries of bilirubin were

12 h at the end of every experiment so as to decrease the adsorption

92.8 ± 1.5% to 104.3 ± 2.2% (n = 5) for intra- and inter-day assays

of bilirubin on them and prolong their life. In a word, all these meas-

at low, medium and high concentrations of bilirubin in the RLM,

ures not only increased stability of the samples, but also improved

HLM and UGT1A1 incubation samples (Table 2). The mean extrac-

accuracy and precision of the analytical method.

tion recoveries of bilirubin (n = 5) from the RLM, HLM and UGT1A1

incubation samples were satisfactory at low, medium and high con-

centrations, which varied from 91.5 ± 1.0% to 104.3 ± 6.7% (Table 2).

3.6. Bilirubin glucuronidation

High recovery of bilirubin from the three incubation systems

suggested that there was negligible loss during the extraction pro-

To guarantee the process of in vitro bilirubin glucuronidation

cedure. Moreover, precision for BMG1, BMG2 and BDG from the

under the initial rate conditions and formation of appropriate

RLM, HLM and UGT1A1 incubation samples at three initial biliru-

amount of bilirubin glucuronides, meanwhile, taking account of

bin concentration levels did not exceed 1.1% of RSD for within-run

the saturation of bilirubin solubility in the incubation solution, as

and 2.9% of RSD for inter-run, respectively. All the data on precision,

well as the maneuverability of the experiment (e.g. rate of biliru-

accuracy and recovery complied with the requirements of bioana-

bin glucuronidation is very quickly), we investigated the incubation

lytical method validation prepared by Center for Drug Evaluation

conditions, e.g. substrate concentration, microsomal or UGT1A1

and Research (CDER) FDA [41].

protein concentration, and incubation time. Finally, biliruin con-

centration ranges of 0.25–2 ␮M, microsomal or UGT1A1 protein

3.5. Stability concentration of 12.5 ␮g/mL, and incubation time of 15 min were

chosen as the optimized incubation conditions to characterize

The stability experiment indicated that bilirubin stock solution bilirubin glucuronidation. The results indicated that, under these

◦

(2 mM, n = 3) was stable at room temperature (25 C) for 6 h and conditions, bilirubin glucuronidation obeyed the Hill equation by

− ◦

70 C for 30 d. Compared to that of the initial time point, the per- RLM, and the Michaelis–Menten equation by HLM and UGT1A1,

centage remaining of bilirubin were ≥98.2 ± 1.8% and 92.2 ± 2.6% respectively. The kinetic proﬁles and parameters of bilirubin glu-

(Table S1 in the supplementary data), respectively. The bilirubin curonidation by RLM, HLM and UGT1A1 were shown in Fig. 3 and

standard solutions were stable at room temperature for 24 h, the Table 4, respectively.

G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159 155

Table 2

Intra- and inter-day precision, accuracy and recovery for the determination of bilirubin in the RLM, HLM and UGT1A1 incubation mixtures, respectively (n = 5, X ± SD).

Assay recovery Extraction recovery

Bilirubin conc. added (␮M) Intra-day Inter-day Recovery RSD

Conc. assayed (␮M) Recovery (%) RSD (%) Conc. assayed (␮M) Recovery (%) RSD %

RLM

0.05 0.049 ± 0.001 98.2 ± 1.4 1.4 0.050 ± 0.001 99.7 ± 1.3 1.3 91.5 ± 1.0 1.1

0.2 0.197 ± 0.004 98.3 ± 1.8 1.8 0.197 ± 0.004 98.5 ± 1.8 1.8 104.3 ± 6.7 6.4

± ±

1.5 1.392 0.022 92.8 1.5 1.6 1.404 0.052 93.6 3.7 3.7 97.6 ± 5.6 5.7

HLM

0.05 0.051 ± 0.0004 101.5 ± 0.8 0.8 0.051 ± 0.001 101.6 ± 2.1 2.1 96.8 ± 0.7 0.8

0.2 0.208 ± 0.001 104.1 ± 0.7 0.6 0.209 ± 0.004 104.3 ± 2.2 2.1 94.7 ± 1.3 1.3

1.5 1.442 ± 0.005 96.1 ± 0.3 0.4 1.444 ± 0.017 96.3 ± 1.2 1.2 94.9 ± 0.7 0.7

UGT1A1

± ±

0.05 0.052 0.001 103.3 1.2 1.2 0.051 0.001 101.3 1.8 1.8 98.3 ± 2.1 2.1

0.2 0.202 ± 0.003 101.2 ± 1.4 1.4 0.204 ± 0.003 102.2 ± 1.6 1.6 97.3 ± 2.0 2.0

1.5 1.438 ± 0.022 95.9 ± 1.5 1.5 1.424 ± 0.015 94.9 ± 1.0 1.1 92.3 ± 0.8 0.9

Conc., concentration(s); RSD, relative standard deviation.

Table 3

◦ ◦ ◦

Stability of bilirubin and its glucuronides in the RLM, HLM and UGT1A1 incubation samples at 37 C, 25 C and −70 C during or after the reaction, respectively (n =3, X ± SD).

Storage condition Incubation system

RLM HLM UGT1A1

a a a a a a a a a

0.2 (␮M) 0.75 (␮M) 1.5 (␮M) 0.2 (␮M) 0.75 (␮M) 1.5 (␮M) 0.2 (␮M) 0.75 (␮M) 1.5 (␮M)

◦

37 C × 15 min

UCB 97.6 ± 2.9 94.1 ± 1.7 99.3 ± 1.0 101.5 ± 3.6 95.8 ± 0.8 97.3 ± 1.3 101.7 ± 4.2 98.9 ± 1.9 99.6 ± 3.3

BMG1 97.5 ± 6.5 98.0 ± 2.6 99.5 ± 5.2 97.6 ± 3.4 100.0 ± 3.5 98.1 ± 3.3 99.1 ± 2.3 99.7 ± 7.8 99.3 ± 2.2

BMG2 102.7 ± 3.0 99.8 ± 3.4 96.9 ± 4.6 99.0 ± 3.0 99.7 ± 1.9 95.7 ± 5.0 98.8 ± 0.9 101.0 ± 1.5 97.3 ± 6.3

± ±

BDG 101.2 3.3 99.1 2.2 98.8 0.9 100.1 5.7 99.9 ± 1.8 98.3 ± 2.4 97.9 ± 4.0 96.5 ± 1.0 99.1 ± 1.8

◦

25 C × 24 h

UCB 87.4 ± 3.6 88.8 ± 0.9 86.6 ± 0.8 100.6 ± 0.2 97.3 ± 1.0 96.7 ± 0.9 95.3 ± 1.8 89.5 ± 2.9 87.0 ± 4.0

BMG1 88.9 ± 5.1 90.4 ± 0.8 85.0 ± 1.7 99.0 ± 1.8 97.4 ± 2.6 94.5 ± 0.5 90.1 ± 8.2 90.9 ± 1.3 90.5 ± 1.2

BMG2 91.2 ± 1.4 85.8 ± 1.0 87.8 ± 0.4 97.4 ± 0.9 95.8 ± 1.0 95.3 ± 0.5 90.0 ± 0.8 90.0 ± 0.4 90.3 ± 0.2

BDG 96.7 ± 1.5 93.9 ± 1.3 94.5 ± 0.8 99.8 ± 0.2 94.6 ± 1.5 96.6 ± 2.7 89.5 ± 0.5 91.0 ± 1.1 94.5 ± 1.1

◦

−70 C × 7 d

b ±

UCB 100.1 2.6 99.3 ± 0.7 100.3 ± 1.5 99.4 ± 1.1 91.4 ± 0.1 87.8 ± 0.9 96.2 ± 3.5 102.0 ± 1.6 96.9 ± 0.6

BMG1 99.8 ± 1.1 100.1 ± 1.7 100.1 ± 0.1 100.5 ± 2.0 98.2 ± 3.2 99.3 ± 0.5 97.4 ± 1.5 100.2 ± 0.4 100.9 ± 0.3

BMG2 100.1 ± 1.3 99.9 ± 0.3 99.6 ± 0.2 99.2 ± 2.1 97.3 ± 5.8 99.8 ± 0.3 101.9 ± 0.3 101.1 ± 0.6 101.4 ± 0.6

BDG 100.0 ± 0.6 102.5 ± 1.1 101.2 ± 2.1 100.9 ± 4.7 97.4 ± 2.2 97.7 ±1.9 100.4 ± 3.9 98.0 ± 2.6 101.2 ± 2.3

SC, storage condition; IS, incubation system.

The concentrations of bilirubin (i.e. UCB) added in the incubation mixtures at the initial reaction time point (0 min).

The values given in the rows are percentage remaining (PR) (%).

As shown in Fig. 3, rank orders of average formation rates RLM, and it showed no signiﬁcant difference (p > 0.05) between

of bilirubin glucuronides were: VUGT1A1 > VRLM > VHLM for BMG1, UGT1A1 and RLM. However, as shown in Table 4, Km and Vmax for

VRLM > VUGT1A1 > VHLM for BMG2, VUGT1A1 > VHLM > VRLM for BDG, and total bilirubin glucuronidation by RLM, HLM, UGT1A1 ranged from

VUGT1A1 ≈ VRLM > VHLM for TBG (p < 0.05), respectively. Under the 0.40 ± 0.022 ␮M to 0.45 ± 0.016 ␮M and 1.86 ± 0.029 nmol/mg/min

same incubation conditions (i.e. the same substrate concentration, to 2.95 ± 0.036 nmol/mg/min in the present study, respectively.

protein concentration and incubation time), the average forma- It is interesting to note that, under the same incubation

tion rates of TBG by HLM were slower than that by UGT1A1 and condition, the apparent kinetic parameters Km, Vmax and CLint

Table 4

Apparent enzyme kinetic parameters of bilirubin glucuronidation by RLM, HLM and UGT1A1, respectively (n = 3, X¯ ± SD).

Incubation systems Kinetic parameters BMG1 BMG2 BDG TBG Kinetic mechanism

␮ ± ± ± ±

RLM Km ( M) 0.45 0.017 0.48 0.019 0.29 0.039 0.45 0.016 Hill equation

Vmax (nmol/mg/min) 0.81 ± 0.019 1.62 ± 0.038 0.19 ± 0.014 2.65 ± 0.057

CLint (mL/mg/min) 1.81 ± 0.060 3.39 ± 0.14 0.58 ± 0.10 5.92 ± 0.22

n 2.17 ± 0.17 1.86 ± 0.12 1.13 ± 0.23 1.85 ± 0.11

HLM Km (␮M) 0.47 ± 0.026 0.48 ± 0.024 0.25 ± 0.026 0.40 ± 0.022 Michaelis–Menten

Vmax (nmol/mg/min) 0.68 ± 0.012 0.86 ± 0.014 0.31 ± 0.007 1.86 ± 0.029 equation

CLint (mL/mg/min) 1.44 ± 0.043 1.80 ± 0.048 1.22 ± 0.058 4.70 ± 0.079

UGT1A1 Km (␮M) 0.66 ± 0.03 0.64 ± 0.027 0.17 ± 0.017 0.44 ± 0.018 Michaelis–Menten

Vmax (nmol/mg/min) 1.16 ± 0.02 1.27 ± 0.020 0.60 ± 0.011 2.95 ± 0.036 equation

CLint (mL/mg/min) 1.75 ± 0.12 2.00 ± 0.084 3.68 ± 0.34 6.72 ± 0.27

TBG = BMG1 + BMG2 + BDG; CLint = Vmax/Km.

156 G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159

RLM HLM UGT1A1 0.8 0.6 0.9 A1 A2 A3

0.6 0.9 0.4 0.6 0.4 0.6 1.0

V 0.5 V 0.3 V 0.5

0.2 0.3

(nmol/mg/min)

0.2 0.0 (nmol/mg/min)

0.0 0.5 1.0 0.0 0.0

0.0 0.5 1.0 1.5 BMG1 formation rate

BMG1 formation rate 0.0 0.5 1.0 1.5

V/C BMG1 formation rate V/C

0 V/C

0 0 0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 [Bilirubin (μM) [Bilirubin (μM) [Bilirubin (μM) 1.6 B1 B2 1 B3 1.2 0.6 0.8 1.0 1.5 0.6 0.8 0.4 1.0 1.0 V V 0.5 0.4 V 0.5 0.5

(nmol/mg/min)

0.4 0.2 (nmol/mg/min)

0.0 (nmol/mg/min) 0.0

0.2 0.0

0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 2.0 BMG2 formation rate 0 1 2 BMG2 formatio rate BMG2 formation rate V/C V/C V/C

0 0

0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2 [Bilirubin (μM) [Bilirubin (μM) [Bilirubin (μM) 0.6 0.2 C1 0.3 C2 C3

0.15 0.4 0.2 0.2 0.6 0.1

0.2 V V 0.1 V 0.3

0.1 0.2

(nmol/mg/min)

0.05 (nmol/mg/min) 0.0

0.0 BDG formation rate

0.0 0 1 2 3 4 BDG formation rate 0.0 0.2 0.4 BDG formation rate 0.0 0.5 1.0 V/C

V/C V/C

0 0

0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2

2.5 1.6 D2 D3 2.5 D1 2 2 1.2 3 2 1.5 3 1.5 2 0.8 2 V V 1 1 V 1 1 1

(nmol/mg/min)

(nmol/mg/min) 0 0.4 0

TBG formation rate 0

0.5 TBG formation rate 0.5 0 1 2 3 TBG formation rate 0 2 4 0 2 4 6 8 V/C V/C V/C

0 0

0 0.5 1 1.5 2 0 0.5 1 1.5 2 0 0.5 1 1.5 2

[Bilirubin (μM) [Bilirubin (μM) [Bilirubin (μM)

Fig. 3. Kinetics proﬁles of bilirubin glucuronidation by RLM (A1–D1), HLM (A2–D2) and UGT1A1 (A3–D3), respectively. BMG1 (A1–A3) and BMG2 (B1–B3) represented two

bilirubin monoglucuronides, and BDG (C1–C3) represented bilirubin diglucuronides. TBG (D1–D3) represented total bilirubin glucuronides, i.e. TBG = BMG1 + BMG2 + BDG.

The Hill equation was ﬁt to the data from the RLM (A1–D1) incubation system, and the Michaelis–Menten equation was ﬁt to the data from the HLM (A2–D2) and UGT1A1

(A3–D3) incubation systems, respectively. The embedded ﬁgures are Eadie–Hofstee plots for the same data. Rhombuses and smooth lines denote the observed and predicted

rates of bilirubin glucuronidation, respectively. Microsomal or UGT1A1 protein concentration was 12.5 ␮g/mL, incubation time was 15 min. Each data point represented the

average of three replicates.

values of bilirubin glucuronidation (i.e. formation of BMG1, conducted using 0.05–200 ␮M bilirubin, 0.05–2.3 mg/mL protein

BMG2, BDG and TBG) exhibited signiﬁcant differences (p < 0.05) from the microsomes or UGT1A1, and 5–35 min incubation time,

in the three different incubation systems (Fig. 4). For example, respectively [26,31–33]. The discrepancy in the present experi-

average kinetic parameter values of TBG formation showed ment and the previous reports is most probably due to different

Km,UGT1A1 ≈ Km,RLM > Km,HLM, Vmax,UGT1A1 > Vmax,RLM > Vmax,HLM incubation conditions selected, e.g. the substrate concentrations,

and CLint,UGT1A1 > CLint,RLM > CLint,HLM. The results indicated that concentrations and sources of UGT1A1 (e.g. different cell lines,

bilirubin had the same binding afﬁnity to the UGT1A1 from the microsomes, supersomes, cDNA-expressed enzymes), reaction

recombinant human UGT1A1 enzyme and RLM, and showed time and assay methodology. Especially, the differences of appar-

the strongest afﬁnity to the UGT1A1 from the HLM. Meanwhile, ent kinetic parameters in our experiment were probably due to

under the same incubation conditions, recombinant human different UGT1A1 source (i.e. pooled rat and human microsomes,

UGT1A1 enzyme demonstrated the strongest capacity and efﬁ- recombinant human UGT1A1 enzyme from BD-Supersomes ),

ciency for bilirubin glucuronidation, HLM is just the reverse. enzymatic activity, and content of UGT1A1 in the RLM, HLM and

In fact, Km and Vmax values of bilirubin glucuronidation were BD-supersomes. Moreover, all the Km values of formation of BMG1

reported to range from 0.20 ␮M to as high as 24 ␮M and 0.08 and BMG2 showed no signiﬁcant difference (p > 0.05) in the same

to 1.08 nmol/mg/min when these incubation experiments were incubation system. This indicated that C8 and C12 carboxyl group

G. Ma et al. / Journal of Pharmaceutical and Biomedical Analysis 92 (2014) 149–159 157

* RLM HLM UGT1A1 RLM 0.8 * * 100 A * 80 0.6 * 60 BG BDG * T BMGs

0.4 of (μM) 40

m * % K 20 0.2

0 0.5 1 1.5 2

0.0

BMG1 BMG2 BDG TBG [Bilirubin] (μM)

HLM bilirubin glucuronides 100

B 3.5 * 80 3.0 * 60 BDG TBG 2.5 of 40 BMGs

2.0 * %

/mg/min) * * 20 ol 1.5

nm *

( 1.0 * 0

max * 0 0.5 1 1.5 2 V 0.5 [Bilirubin] (μM)

0.0

UGT1A1

BMG1 BMG2 BDG TBG 100

bili rubin glucuronides 80 * 7.0 C * 60 BDG TBG 6.0

of BMGs 40 %

) 5.0 *

in * 20 4.0 * *

3.0 0

* 0 0.5 1 1.5 2 (mL/mg/m *

2.0 int [Bilirubin] (μM)

CL 1.0

Fig. 5. Proportions of BMGs and BDG formed in the RLM, HLM and UGT1A1 incuba-

0.0 tion systems, respectively. BMGs = BMG1 + BMG2; microsomal (or UGT1A1) protein

BMG1 BMG2 BDG TBG concentration 12.5 ␮g/mL; incubation time 15 min.

bili rubin glucuronides

It needs to be pointed out that it looked the glucuronidation

Fig. 4. Comparison of kinetic parameters of bilirubin glucuronidation by RLM, HLM reaction of biliruin was not reach Vmax in some conditions (e.g. A2,

and UGT1A1, respectively. A, B and C were the corresponding column plots sum-

B2, A3, B3, etc.) in Fig. 3. This is probably related with the substrate

marizing the values of Km, Vmax and CLint, respectively. “*”, signiﬁcant difference

(i.e. bilirubin) concentration selected. The maximum bilirubin con-

(p < 0.05). Microsomal (or UGT1A1) protein concentration 12.5 ␮g/mL; incubation

centration selected was 2 ␮M in the study, which was based on

time 15 min.

the insolubility of bilirubin in the incubation solutions and poor

linearities of bilirubin standard solution when its concentration

was >2 ␮M. Taking account of the saturation of bilirubin solubil-

of bilirubin have the same afﬁnity to UGT1A1 when UCB was ity in the incubation solutions, it was not appropriate to further

glucuronidated to BMGs (i.e. BMG1 and BMG2) by RLM (HLM or increase bilirubin concentration to >2 ␮M. In fact, as shown in

UGT1A1). The apparent Vmax values of formation of BMG1 and Fig. 3, the observed and predicted rates of bilirubin glucuronida-

BMG2 showed no signiﬁcant difference (p > 0.05) in the UGT1A1 tion showed excellent goodness of ﬁt. The Eadie–Hofstee plots, the

incubation system, but Vmax,BMG2 > Vmax,BMG1 (p < 0.05) in the RLM residual sum of squares (R ≥ 0.98) and Akaike information crite-

and HLM incubation system. This indicated that recombinant rion (AIC ≤ −112.97) also exhibited excellent ﬁtting for the data

human UGT1A1 enzyme has the same glucuronidation capacity and model. It indicated that apparent enzyme kinetic parameters

for formation of BMG1 and BMG2, but RLM and HLM have stronger of bilirubin glucuronidation were reliable in the tested substrate

glucuronidation capacity for formation of BMG2 than that of BMG1. concentration range selected.

CLint,BMG2 > CLint,BMG1 (p < 0.05) in the three incubation systems In addition, our results indicated that proportions of BMGs and

indicated that efﬁciency of intrinsic clearance of BMG2 was higher BDG formed depended on bilirubin concentration and enzyme

than that of BMG1. sources. As shown in Fig. 5, BMGs were the dominant species

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Appendix A. Supplementary data

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