Chemosphere 133 (2015) 6–12
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Chemosphere
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Urinary bromophenol glucuronide and sulfate conjugates: Potential
human exposure molecular markers for polybrominated diphenyl ethers
Ka-Lok Ho a, Man-Shan Yau a, Margaret B. Murphy a,⇑, Yi Wan b,1, Bonnie M.-W. Fong c,d, Sidney Tam c,
John P. Giesy a,b,e,f,g,h, Kelvin S.-Y. Leung d, Michael H.-W. Lam a,⇑
a
State Key Laboratory for Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Hong Kong Special Administrative Region
Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Canada
Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong Special Administrative Region
d
Department of Chemistry, Hong Kong Baptist University, Hong Kong Special Administrative Region
e
Department of Zoology and Center for Integrative Toxicology, Michigan State University, USA
f
School of Biological Sciences, The University of Hong Kong, Hong Kong Special Administrative Region
g
Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
h
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China
b
c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Parallel blood and urine samples were
collected from 100 donors in Hong
Kong.
Levels of selected BP glucuronide and
sulfate conjugates in urine were
determined.
Their levels were found to correlate
well with that of PBDEs in blood
plasma.
Our results suggest that BP
conjugates can be useful markers for
PBDE exposure.
a r t i c l e
i n f o
Article history:
Received 10 October 2014
Received in revised form 18 February 2015
Accepted 1 March 2015
Available online 24 March 2015
Handling Editor: Andreas Sjodin
Keywords:
Bromophenols
Polybrominated diphenyl ethers
Metabolites
Exposure molecular markers
a b s t r a c t
One possible source of urinary bromophenol (BP) glucuronide and sulfate conjugates in mammalian animal models and humans is polybromodiphenyl ethers (PBDEs), a group of additive flame-retardants
found ubiquitously in the environment. In order to study the correlation between levels of PBDEs in
human blood plasma and those of the corresponding BP-conjugates in human urine, concentrations of
17 BDE congeners, 22 OH-BDE and 13 MeO-BDE metabolites, and 3 BPs in plasma collected from 100
voluntary donors in Hong Kong were measured by gas chromatograph tandem mass spectrometry
(GC–MS). Geometric mean concentration of RPBDEs, ROH-BDEs, RMeO-BDEs and RBPs in human plasma
were 4.45 ng g1 lw, 1.88 ng g1 lw, 0.42 ng g1 lw and 1.59 ng g1 lw respectively. Concentrations of glucuronide and sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) in
paired samples of urine were determined by liquid chromatography tandem triple quadrupole mass
spectrometry (LC–MS/MS). BP-conjugates were found in all of the parallel urine samples, in the range
of 0.08–106.49 lg g1-creatinine. Correlations among plasma concentrations of RPBDEs/ROH-BDEs/
⇑ Corresponding authors at: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong Special Administrative Region. Tel.:
+852 3442 6888; fax: +852 3442 7406 (M.B. Murphy). Department of Biology & Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong Special
Administrative Region. Tel.: +852 3442 7329; fax: +852 3442 0522 (M.H.-W. Lam).
E-mail addresses: mbmurphy@cityu.edu.hk (M.B. Murphy), bhmhwlam@cityu.edu.hk (M.H.-W. Lam).
1
Present address: Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing, People’s Republic of China.
http://dx.doi.org/10.1016/j.chemosphere.2015.03.003
0045-6535/Ó 2015 Elsevier Ltd. All rights reserved.
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
Human blood plasma
Human urine
7
RMeO-BDEs/RBPs and BP-conjugates in urine were evaluated by multivariate regression and Pearson
product correlation analyses. These urinary BP-conjugates were positively correlated with RPBDEs in
blood plasma, but were either not or negatively correlated with other organobromine compounds in
blood plasma. Stronger correlations (Pearson’s r as great as 0.881) were observed between concentrations
of BDE congeners having the same number and pattern of bromine substitution on their phenyl rings in
blood plasma and their corresponding BP-conjugates in urine.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Polybrominated diphenyl ethers (PBDEs), a class of brominated
flame retardants (BFRs), have aroused considerable public concern
because of their resistance to environmental degradation, especially for lower brominated congeners, their tendency to
bioaccumulate and potential adverse effects on health of humans
(de Boer et al., 2000; Hooper and McDonald, 2000; Alaee et al.,
2003; Guvenius et al., 2003; Henrik and Birger, 2010). In 2009,
the Penta- and Octa-BDE commercial mixtures were listed as persistent organic pollutants (POPs) under the Stockholm Convention
(Eljarrat and Barceló, 2011). Despite international efforts on the
restriction of their production and usage, PBDEs are likely to
remain in the global ecosystem for a considerable period of time
because of their slow rate of degradation for lower brominated
congeners, and the fact that large amounts of manufactured goods
containing PBDEs are still in use (Harrad et al., 2006; Betts, 2008).
Thus, the continuous monitoring of accumulation of PBDEs in
humans is still important for the accurate assessment of their risk
to public health at national and international levels.
The most frequently adopted approach to monitor human exposure to PBDEs is the direct quantification of selected BDE congeners
and their hydroxylated (OH-BDEs) and methoxylated (MeO-BDEs)
species in blood/serum (Athanasiadou et al., 2008; Turyk et al.,
2008; Roosens et al., 2009; Wang et al., 2012), and human breast
milk (Sudaryanto et al., 2008; Schuhmacher et al., 2009, 2013;
Toms et al., 2009; Shi et al., 2013). Other human tissues such as
hair, kidney, lung, liver and adipose tissues have also been used
(Covaci et al., 2008; Zhao et al., 2008, 2009; Zheng et al., 2014).
However, collecting human tissue samples from people for chemical/biochemical analysis and risk assessment is an intrusive operation and difficult to achieve in large-scale population-wide or
national surveys. While sampling of human hair and breast milk
can be considered non-intrusive processes, they face other limitations, such as the ease of exogenous contamination of hair samples (Morris et al., 2012; Barbosa et al., 2013) and the gender and
age distribution restrictions of sampling breast milk (Landrigan
et al., 2002). Alternatively, sampling of human urine is simple,
quick and non-intrusive, making it much easier to obtain urine
samples from a large number of voluntary donors within a community for large-scale surveys.
Occurrence of metabolites of selected BDE congeners in urine of
mammalian animal models has already been well established in
several toxicokinetic studies (Hakk and Letcher, 2003; Chen et al.,
2006; Sanders et al., 2006). These metabolites are mainly glucuronide and sulfate conjugates of dibromophenols (DBPs) and tribromophenols (TBPs), probably because of their lower molecular
weight (relative to their parent BDE congeners) that facilitates
their renal removal. Our research team has previously reported
the synthesis, purification, and characterization of glucuronide
and sulfate conjugates of bromophenols (BP) and has developed
an analytical protocol for their determination in human urine
(Ho et al., 2012). A preliminary survey on 20 voluntary donors
revealed the presence of at least one of these BP-conjugates in
their urine. In this study, we examined the correlation between
concentrations of PBDEs/OH-BDEs/MeO-BDEs/BPs and in blood
plasma and BP-glucuronide and -sulfate conjugates in urine of
humans. A total of 100 matched samples of plasma and urine were
collected from volunteer donors in Hong Kong, China. The objective
of this work was to evaluate whether glucuronide and sulfate conjugates of BPs in human urine are suitable molecular markers for
the assessment of population exposure to PBDEs.
2. Experimental
2.1. Safety precautions
All necessary precautions were taken during the handling of
samples of blood and urine. Double latex gloves, facemasks and
eye-protection goggles were worn at all times during handling,
spiking and transfer of samples from humans. All spent samples
of urine were collected after analysis in separate capped containers
with proper clinical waste labels. Both spent samples and used
personal protection items were treated as clinical waste and
were collected and disposed of in accordance with the ‘‘Code of
Practice for the Management of Clinical Waste’’ issued by the
Environmental Protection Department of the Hong Kong SAR
Government.
2.2. Sample collection
All studies that involved human tissues and body fluids were
conducted in accordance with guidelines of the Research Ethics
Committee of City University of Hong Kong after proper approvals
were given by the Committee. Parallel samples of human plasma
and urine (n = 100; 50 from male and 50 from female donors) were
collected from voluntary donors during March to July 2010 by
registered doctors and nurses at Queen Mary Hospital, Hong
Kong. Besides their gender and age, no other personal information
of those voluntary donors was collected. The age range of the
volunteers was from 16 to 93 years of age (mean ± SD:
54.9 ± 21.9 years). These donors were subdivided into different
age groups for comparison: age 16–25 (n = 11); age 26–35
(n = 15); age 36–45 (n = 12); age 46–55 (n = 14); age 56–65
(n = 15); age 66–80 (n = 13) and age > 80 (n = 20).
Samples of whole blood were collected using the standard phlebotomy technique in vacutainer tubes containing sodium heparin
anticoagulant (Vacuette, Greiner bio-one, GmbH, Austria). Whole
blood was then centrifuged at 1500g for 25 min. Plasma was
removed from the top of the tube. Morning-first urine samples
were collected in 100 mL sterilized glass bottles and stored at
80 °C within 15 min of sampling until analysis. Urine from each
donor was subdivided into three replicate samples before lowtemperature storage. All samples were carefully labeled and
documented. Upon analysis, samples were thawed, and 10 mL of
each sample was retained for creatinine content determination
(D’Haese et al., 1985). Creatinine determination was conducted
by a kinetic colorimetric assay based on the modified Jaffe method
using the Roche Modular System (Roche Diagnostics, IN, USA), with
an analytical range between 360 and 57 500 mmol L1.
8
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
2.3. Sample extraction and preparation
Synthesis, purification and characterization of the glucuronide
and sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6tribromophenol (2,4,6-TBP), and the analytical protocols for the
quantification of PBDEs, OH-BDEs, MeO-BDEs and BPs in human
plasma and the BP-conjugates in human urine have been described
in our previous publications (Hovander et al., 2000; Qiu et al.,
2009; Ho et al., 2012). Details on materials, instrumentation,
extraction methods, and QA/QC protocols of this study are given
in the Supporting Information.
2.4. Analysis of data
All statistical analyses were performed using SPSS 16 (SPSS Inc.,
Chicago, IL), Prism 2.01 (GraphPad Software, Inc.) and Sigmastat
3.5 (Sigmastat, Jandel Scientific, CA). Normality of data was
checked by the Klomogorov-Smirnov test. Logarithm, natural-logarithm, arcsine, square root, reciprocal square root or cubic root
transformations were used whenever fit to obtain normally
distributed data sets for parametric statistical testing. Student’s
t-test was used to compare concentrations of PBDEs, MeO-BDEs,
OH-BDEs and BPs, in human plasma samples, and BP-conjugates,
in human urine samples, between male and female donors. If data
were not normally distributed, a non-parametric Mann–Whitney
Rank Sum test was used for the comparison. One-way ANOVA
(parametric) or ANOVA on Ranks (non-parametric) tests were used
to compare concentrations of the target brominated species in
plasma and urine among different age categories. Multivariate
linear regression and Pearson product moment correlation analysis
were used to examine the influence of concentrations of PBDEs,
MeO-BDEs, OH-BDEs, BPs in blood plasma on concentrations of
BP-conjugates in urine. A P < 0.05 was considered statistically
significant for all statistical measurements.
3. Results and discussion
3.1. Concentrations of PBDEs, OMe-PBDEs, OH-PBDEs and BPs in
human plasma
Concentrations of RPBDEs, RMeO-PBDEs, ROH-PBDEs and
RBPs in plasma, normalized by the plasma lipid weight (lw), are
summarized (Table 1). PBDEs were detected in all of the 100
human plasma samples, with RPBDEs ranging from 0.01 to
18.2 ng g1 lw. These concentrations of RPBDEs are quite
comparable to those reported in previous studies in other Asian
countries (Tan et al., 2008; Zhu et al., 2009; Uemura et al., 2010;
Kim et al., 2012), New Zealand (Harrad and Porter, 2007) and some
European countries (Thomas et al., 2006; Gómara et al., 2007;
Antignac et al., 2009; Kalantzi et al., 2011), but are lesser than
those in the population of Northern America (Schecter et al.,
2005; Sandanger et al., 2007; Lunder et al., 2010) and those who
live near sites where BFR are produced or used in large quantities
(Jin et al., 2009), or e-waste disposal/dismantling areas (Bi
et al., 2007) (Fig. S1, Supporting Information). Similar to many
previous studies on the occurrence of PBDEs in human plasma,
BDE-47, 99 and 209 were the most abundant BDE congeners
detected in this study, with geometric mean concentrations of
0.55 ng g1 lw; (95% confidence interval: 0.39–0.79 ng g1 lw),
0.33 ng g1 lw (95% confidence interval: 0.23–0.48 ng g1 lw), and
0.47 ng g1 lw (95% confidence interval: 0.35–0.62 ng g1 lw),
respectively. There was no significant difference between concentrations of PBDEs in plasma among all age categories (p = 0.736)
or gender (p = 0.143). BDE-47 and 99 were the two most frequently detected congeners with a detection frequency of >90%,
while <20% of the plasma samples contained BDE-209. This frequency of occurrence of BDE-209 is much less compared to surveys
carried out in the US, European countries and Japan. A recent study
by Wang et al. (2011) reported greater concentrations and occurrence frequencies of BDE-47 and 99 in seafood from fish markets
in Hong Kong. This suggests that food consumption rather than
inhalation of indoor dust, an environmental matrix with greater
concentrations of BDE-209, might be a more significant exposure
route to the residents of Hong Kong.
OH-BDEs and BPs were detected in human blood at concentrations similar to those of PBDEs. This is consistent with findings of
previous studies (Athanasiadou et al., 2008; Roosens et al., 2009;
Qiu et al., 2009; Wan et al., 2010). 6-OH-BDE-47 and 50 -OH-BDE99 were the two most abundant OH-BDE congeners in the human
blood plasma samples, with geometric mean concentrations of
0.32 ng g1 lw; (95% confidence interval: 0.23–0.45 ng g1 lw)
and 0.06 ng g1 lw (95% confidence interval: 0.03–0.11 ng g1 lw),
respectively. Three different BPs, namely 2,4-DBP, 2,4,5-tribromophenol (2,4,5-TBP) and 2,4,6-TBP, were detected in 80% of the
plasma samples. The geometric mean concentration of RBPs was
1.59 ng g1 lw (95% confidence interval: 1.34–1.89 ng g1 lw). The
occurrence of these three BPs in mouse plasma after exposure to
the commercial Penta-BDE mixture DE-71 has been reported by
Qiu et al. (2007). In an in vitro study of the metabolism of BDE99 by human hepatocytes, Stapleton et al. (2009) also determined
2,4,5-TBP in the cell extracts, which was deemed to be generated
by the catabolic cleavage of the diphenyl ether linkage of the
BDE congener. These studies suggest that the diphenyl ether cleavage of PBDEs constitutes one of the sources of BPs (including 2,4DBP and 2,4,6-TBP) in blood plasma. Other sources may include
food consumption and exposure to other BFRs. 2,4-DBP and
2,4,6-TBP have been detected in fresh fish samples commonly consumed in Hong Kong (Chung et al., 2003). 2,4,6-TBP has been used
as a flame retardant, and population may be exposed to it in a
similar way as to PBDEs. There was no significant difference in
the concentrations of OH-BDEs and BPs in human plasma between
male and female donors (p = 0.749 for OH-BDEs, p = 0.165 for BPs).
There were also no significant differences in OH-BDE and BP
content in human plasma samples among all the age groups
(p = 0.449 for OH-BDEs, p = 0.571 for BPs).
MeO-BDEs were also found in human plasma samples. The geometric mean concentration of RMeO-BDEs was 0.42 ng g1 lw (95%
confidence interval: 0.31–0.56 ng g1 lw). This concentration is
comparable to that revealed in a previous study carried out in
Hong Kong (Wang et al., 2012). 6-MeO-BDE-47 and 4-MeO-BDE17 were the two most abundant MeO-BDE congeners, with mean
concentrations in human plasma of 0.88 and 0.73 ng g1 lw,
respectively. This pattern of occurrence of MeO-BDEs in human
plasma samples is different from that observed in the US (Qiu
et al., 2009), perhaps because some abundant MeO-BDE congeners
(6-MeO-BDE-47, 4-MeO-BDE-17, 2-MeO-BDE-28) were not
included in the previous study. There were no significant differences among concentrations of MeO-BDEs in human plasma
among all the age groups (p = 0.210), and no statistically significant
differences in the concentrations of MeO-BDEs were found
between male and female donors (p = 0.702).
3.2. Concentrations of BP-glucuronide and -sulfate conjugates in
human urine samples
To the best of our knowledge, BP-glucuronide and –sulfate conjugates are not commercially available. Authentic standards of the
glucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP for
LC–MS/MS determination in this work were obtained via chemical
synthesis and liquid chromatographic purification. On the other
hand, the presence of conjugates of 2,4,5-TBP in urine samples
9
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
Table 1
Concentrations (ng g1 lw) of RPBDEs, RMeO-BDEs, ROH-BDEs, and RBPs in human plasma samples (n = 100) from Hong Kong, China.
Congeners
Human plasma samples
Total (n = 100)
RPBDEs
RMeO-BDEs
ROH-BDEs
RBPs
Male (n = 50)
Female (n = 50)
GMa (95% CI)b
Min–max
% of detection
GMa (95% CI)b
Min–max
% of detection
GMa (95% CI)b
Min–max
% of detection
4.45
0.42
1.88
1.59
0.01–18.20
N.D.–6.87
N.D.–8.88
N.D.–7.18
100
89
83
84
5.13
0.44
1.82
1.79
0.12–18.20
N.D.–5.15
N.D.–5.19
N.D.–5.16
100
90
86
92
3.82
0.40
1.94
1.40
0.01–14.18
N.D.–6.87
N.D.–8.88
N.D.–7.18
100
90
80
78
(3.66–5.41)
(0.31–0.56)
(1.53–2.29)
(1.34–1.89)
(4.10–6.41)
(0.30–0.65)
(1.40–2.36)
(1.43–2.25)
(2.77–5.28)
0.25–0.63
(1.43–2.64)
(1.08–3.00)
N.D.: not detected.
a
GM: geometric mean.
b
CI: confidence interval.
was not determined in this study because of the unavailability of
2,4,5-TBP starting material for the synthesis of its conjugates.
One or more of the 2,4-DBP- and 2,4,6-TBP-glucuronide and –
sulfate conjugates were detected in all of the urine samples, with
RBP-conjugates ranging from 0.08 to 106.49 lg g1-creatinine
(Table 2). Conjugates of 2,4,6-TBP were the most frequently
detected, while the frequency of detection of 2,4-DBP conjugates
was around 70%. BP-glucuronides were 5- to 10-fold more
abundant than their corresponding BP-sulfates. There were no statistically significant differences in concentrations of BP-conjugates
in urine among age groups (p = 0.378 for 2,4-DBP-sulfate; p = 0.558
for 2,4,6-TBP-sulfate; p = 0.25 for 2,4-DBP-glucuronide; and p
= 0.976 for 2,4,6-TBP-glucuronide). Alternatively, a statistically
significant difference was observed in the urine concentrations
of 2,4-DBP-glucuronide between male and female donors
(p = 0.038), with concentrations of 2,4-DBP-glucuronide found to
be greater in men. A previous study on human urinary bisphenol
A (BPA) in Korea also found a similar phenomenon where concentrations of BPA-glucuronides in men were greater than those in
women.
3.3. Correlations between BP-glucuronide and -sulfate conjugates and
RPBDEs, ROMe-PBDEs, ROH-PBDEs and RBPs
Multivariate linear regression analysis was employed to explore
the relationship among different forms of BDEs/BPs in blood
plasma and BP-conjugates in urine. The standardized regression
coefficient b was used to quantify the relationship between BPconjugates and the various determinants. The first regression
model was built using the natural log-transformed summation of
concentrations of PBDEs, OH-BDEs, MeO-BDEs and BPs, i.e.
lnRPBDEs, lnROH-PBDEs, lnRMeO-PBDEs and lnRBPs, in plasma
samples as independent variables, and the sum of all four BPglucuronide and -sulfate conjugates, i.e. lnRBP-conjugates, in urine
samples as the dependent variable (Table 3). The coefficient of
determination (R2) of the regression model established was
only 0.223, and the model was significantly affected by both
lnRPBDEs and lnRBPs, with their standardized regression coefficients (b) being 0.716 and 0.585 respectively (p < 0.001 for
lnRPBDEs and p = 0.013 for lnRBPs).
While there might not be simple relation between total concentrations of PBDEs in human blood and that of total BP-conjugates in
human urine, we explored correlations among structurally-related
organobromine species in blood and in urine. Based on numerous
previous in vivo studies on laboratory mice and in vitro studies
on human cells, catabolic cleavage of PBDEs at the ether linkage
can produce water-soluble glucuronide and sulfate BP-conjugates
with the number and relative position of the bromine-substituents
resembling that of the corresponding parent BDE congeners.
Thus, correlations between concentrations of PBDEs/MeO-BDEs/
OH-BDEs/BP with 2,4-dibromo and 2,4,6-tribromo substitution in
the blood plasma samples and the glucuronide and sulfate conjugates of 2,4-DBP/2,4,6-TBP in urine were investigated (Tables 4a
and 4b). The R2 values increased to 0.744 (lnR2,4-DBP-conjugates)
and 0.707 (lnR2,4,6-TBP-conjugates), which suggests that the
refined regression models better explained the variability of the
dependents. They also gave better standardized coefficients (b):
4.34 for lnR2,4-dibromo-BDEs and 3.23 for lnR2,4,6-tribromoBDEs, revealing much stronger relationships between R2,4-dibromo-BDEs/R2,4,6-tribromo-BDEs in human plasma and their
corresponding BP-conjugates in urine. All the variance inflation
factors (VIFs) of the independent variables were <2, indicating
the absence of multicollinearity in the regression models. Thus,
our results revealed strong relationships between BP-glucuronide
and -sulfate conjugates in human urine and R2,4-dibromo-BDEs
and R2,4,6-tribromo-BDEs in human blood plasma. On the other
hand, their relationships with RMeO-BDEs, ROH-BDEs, or RBPs
in blood plasma were of less significance. These results suggest
that urinary BP-conjugates in human originated from exposure to
PBDEs rather than MeO-BDEs, OH-BDEs or BPs.
Pearson product moment correlation was used to evaluate the
correlations between natural-log-transformed RBP-conjugates
and RPBDEs, RMeO-BDEs, ROH-BDEs and RBPs. Strong relationships were observed between lnR2,4-dibromo-BDEs in human
blood vs lnR2,4-DBP-conjugates in human urine and lnR2,4,6-tribromo-BDEs in human blood vs lnR2,4,6-TBP-conjugates in human
urine (Pearson’s r = 0.881 and 0.823, respectively; Tables 5a and
5b). These results demonstrate the correlation between urinary
concentration of BP-glucuronide and -sulfate conjugates and the
level of PBDEs in blood plasma (Figs. 1 and 2). On the other hand,
concentrations of urinary BP-glucuronide and -sulfate conjugates
do not correlate well with those of lnRMeO-BDEs, lnROH-BDEs
and lnRBPs in blood plasma. This suggests that the glucuronide
and sulfate conjugates of BPs in human urine may be useful as
molecular markers for human exposure to PBDEs. It is arguable
that these BP-conjugates may only be able to reflect human exposure to PentaBDEs, but not OctaBDEs and DecaBDE. The apparent
half-life of OctaBDEs and DecaBDE in human serum are less than
91 days (Thuresson et al., 2006), which is much shorter than that
of 2 years for PentaBDEs (Geyer et al., 2004). Thus, even though
the bromophenol conjugates may be more related to PentaBDEs,
they can still be useful in revealing long term exposure to PBDEs.
Owing to the unavailability of 2,4,5-TBP, correlation between
R2,4,5-tribromo-BDEs in human blood and R2,4,5-TBP-conjugates
in human urine was not explored in this study. Also, the presence
of BDE-glucuronide and -sulfate conjugates in human urine was
not determined because of the unsufficient quantity of the
corresponding OH-BDEs available for the chemical synthesis of
the metabolites. Their aptness as molecular markers for population
exposure to PBDEs will have to be addressed in the future. Infants
and children were not included in this study because of ethical
considerations associated with the collection of their blood and
10
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
Table 2
Concentrations (lg g1 creatinine) of BP-glucuronide and -sulfate conjugates in human urine samples (n = 100) from Hong Kong, China.
Compound
Samples of human urine
Total (n = 100)
2,4-DBP glucuronide
2,4-DBP sulfate
2,4,6-TBP glucuronide
2,4,6-TBP sulfate
Male (n = 50)
Female (n = 50)
GMa (95% CI)b
Min–max
% of detection GMa (95% CI)b
Min–max
% of detection GMa (95% CI)b
Min–max
% of detection
0.32
0.11
0.87
0.10
N.D.–23.81
N.D.–2.08
N.D.–102.21
N.D.–2.93
71
86
68
94
0.01–23.81
N.D.–2.08
N.D.–102.21
N.D.–2.93
76
88
54
94
N.D.–7.52
N.D.–2.08
N.D.–44.08
N.D.–2.93
66
84
82
94
(0.23–0.44)
(0.08–0.14)
(0.58–1.30)
(0.08–0.13)
0.42
0.10
0.98
0.10
0.27–0.64
(0.07–0.15)
(0.51–1.73)
(0.07–0.15)
0.21
0.11
0.80
0.10
(0.13–0.34)
(0.08–0.17)
(0.47–1.38)
(0.07–0.14)
N.D.: not detected.
a
GM: geometric mean.
b
CI: confidence interval.
Table 3
Significant independent variables of urinary concentrations of BP-conjugates revealed
by multivariate linear regression analysis.
R2
Model summary
0.223
Independent variable
ba
Pb
lnRPBDEs
lnRMeO-BDEs
lnROH-BDEs
lnRBPs
0.716
0.063
0.23
0.585
<0.001
0.632
0.239
0.013
Table 4b
Significant independent variables of urinary concentrations of 2,4,6-TBP-conjugates
revealed by multivariate linear regression analysis.
R2
Model summary
0.707
VIFc
Independent variable
ba
Pb
VIFc
1.056
1.059
1.041
1.056
lnR2,4,6-Tribromo-BDEs
lnR2,4,6-TBP
3.226
0.135
<0.001
0.428
1.005
1005
Dependent value was urinary lnR[BP-conjugates].
b: Standardized regression coefficients, slope from the analysis of the model
regression of lnRBP-conjugates versus independent variables.
b
P-value for the term in the multiple linear regression, P < 0.05, statistically
significant.
c
VIF: variance inflation factor.
a
urine samples. However, previous studies have revealed an
inverted age-dependent accumulation of PBDEs, perhaps because
of the dietary preferences, greater frequency of hand-to-month
activities and greater metabolic rate of children (Fischer et al.,
2006; Lunder et al., 2010; Eskenazi et al., 2011; Gari and Grimalt,
2013). Thus, it is worthy to further explore the correlation between
plasma PBDEs and urinary BP-conjugates in children. Another area
that needs further study is potential ethnic differences in the efficacy of PBDE metabolism. Previous studies have shown that
greater glucuronidation of morphine occurred in Chinese people
compared to Caucasians (Zhou et al., 1993). Alternatively, ethnic
Chinese were less able to metabolize codeine by glucuronidation
(Yue et al., 1989, 1991). Results from Goldzieher and coworker
have shown that the pattern of glucuronide conjugation as well
as oxidative metabolism of estrogens (ethinyl estradiol) differed
among Nigerian, Sri Lankan and American populations (Williams
and Goldzieher, 1980; Goldzieher and Brody, 1990). Another study
of the excretion of N-glucuronide conjugates of nicotine and
Table 4a
Significant independent variables of urinary concentrations of 2,4-DBP-conjugates
revealed by multivariate linear regression analysis.
R2
Model summary
0.744
Independent variable
ba
Pb
VIFc
lnR2,4-Dibromo-BDEs
lnR2,4-Dibromo-MeO-BDEs
lnR2,4-Dibromo-OH-BDEs
lnR2,4-DBP
4.34
0.0138
0.0682
0.0345
<0.001
0.834
0.547
0.617
1.089
1.071
1.001
1.059
Dependent value was urinary lnR[2,4-DBP-conjugates].
b: Standardized regression coefficients, slope from the analysis of the model
regression of lnR2,4-DBP-conjugates versus independent variables.
b
P-value for the term in the multiple linear regression, P < 0.05, statistically
significant;
c
VIF: variance inflation factor.
a
Table 5a
Correlation coefficients between urinary 2,4-DBP-conjugates and the various PBDEs/
MeO-BDEs/OH-BDEs/BPs in human plasma.
Total (n = 100) Male (n = 50)
Female
(n = 50)
r
r
P
r
P
P
lnR2,4-Dibromo-BDEs
0.881 <0.05
0.871 <0.05
0.884 <0.05
lnR2,4-Dibromo-MeO-BDEs 0.080 0.449 0.047 0.77 0.100 0.526
lnR2,4-Dibromo-OH-BDEs 0.095 0.452 0.053 0.743 0.200 0.221
lnR2,4-DBP
0.157 0.187 0.223 0.184 0.102 0.561
Analysis of urinary BP conjugates were conducted after natural-log transformation.
Table 5b
Correlation coefficients between urinary 2,4,6-TBP-conjugates and the various
congeners PBDEs and BPs in human plasma.
Total (n = 100)
Male (n = 50)
Female (n = 50)
r
r
r
P
P
P
lnR2,4,6-Tribromo-BDEs
0.823 <0.05
0.820 <0.05
0.834 <0.05
lnR2,4,6-TBP
0.007 0.907 0.06
0.699 0.074 0.655
Analysis of urinary BP conjugates were conducted after natural-log transformation.
Dependent value was urinary lnR[2,4-DBP-conjugates].
a
b: Standardized regression coefficients, slope from the analysis of the model
regression of lnR2,4-DBP-conjugates versus independent variables.
b
P-value for the term in the multiple linear regression, P < 0.05, statistically
significant;
c
VIF: variance inflation factor.
cotinine has shown that people with African origins excreted significantly less glucuronide conjugates than Caucasians (Caraballo
et al., 1998; Benowitz et al., 1999). Information on the differences
in the metabolism of POPs among ethnic groups is scarce. Matters
are further complicated by the differences in the compositions of
PBDE mixtures used in different parts of the world, and dietary
habits of people. Zota and coworkers (Zota et al., 2008) have shown
that PBDE exposure at different regions of the US was different.
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
Fig. 1. Correlation analyses between the concentration of natural logarithmtransformed R2,4-dibromo-BDEs in human plasma and natural logarithmtransformed urinary R2,4-DBP-conjugates.
Fig. 2. Correlation analyses between the concentration of natural logarithmtransformed R2,4,6-tribromo-BDEs in human plasma and natural logarithmtransformed urinary R2,4,6-TBP-conjugates.
Acknowledgements
This work is support by a grant from Research Grants Council of
the Hong Kong Special Administrative Region, China [Reference No.
CityU 9041623]. Prof. Giesy was supported by the program of 2012
‘‘Great Concentration Foreign Experts’’ (#GDW20123200120)
funded by the State Administration of Foreign Experts Affairs, the
P.R. China to Nanjing University and the Einstein Professor
Program of the Chinese Academy of Sciences. He was also supported by the Canada Research Chair program, a Visiting
Distinguished Professorship in the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, City
University of Hong Kong.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chemosphere.
2015.03.003.
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1
2
Urinary Bromophenol Glucuronide and Sulfate Conjugates: Potential Human
3
Exposure Molecular Markers for Polybrominated Diphenyl Ethers
4
5
Ka-Lok Ho,a Man-Shan Yau. a Margaret B. Murphy, *a Yi Wan,†b Bonnie M. –W. Fong,c,d Sidney Tam,c
6
John P. Giesy,a,b,,e,f,g,h Kelvin S. –Y. Leung,d Michael H. –W. Lam*a
7
8
9
10
11
Supporting Information
12
13
14
15
16
17
18
Page
20
1
19
21
Table of Content
22
Methods
23
Materials and General Procedures
24
Instrumentation
25
Identification and Quantification
26
Quality Assurance and Quality Control
27
28
29
Figures
Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various
30
countries.
31
2
References
Page
32
33
Methods
34
Materials and General Procedures.
35
All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as
36
received unless stated otherwise. Oasis WAX® (6 mL / 150 mg) and HLB® (6 mL / 200 mg)
37
cartridges were obtained from Waters Corp. (Milford, MA, USA). Dihexylammonium acetate
38
(DHAA) was obtained from Sigma-Aldrich. Standards for polybrominated diphenylethers
39
(PBDE), including BDE-3, BDE-15, BDE-28, BDE-47, BDE-66, BDE-85, BDE-99, BDE-100,
40
BDE-153, BDE-154, BDE-183, BDE-184, BDE-197, BDE-202, BDE-207, BDE-208, BDE-209;
41
recovery spike standard (13C12-BDE-77 and
42
and
43
standards for bromophenols (BP), 2,4-dibromophenol (2,4-DBP), 2,4,5-tribromophenol
44
(2,4,5-TBP) and 2,4,6-tribromophenol (2,4,6-TBP), and five hydroxylated-PBDE (OH-BDE)
45
(4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 5’-OH-BDE-99 and 6’-OH-BDE-99) standards
46
were purchased from Accustandard (New Haven, Connecticut, USA). All other OH-BDEs,
47
MeO-BDEs (2’-OH-BDE-7, 3’-OH-BDE-7, 4’-OH-BDE-17, 6’-OH-BDE-17, 2’-OH-BDE-28,
48
6-OH-BDE-47,
4’-OH-BDE-49,
49
4-OH-BDE-90,
6-OH-BDE-90,
50
2-OH-BDE-123,
6-OH-BDE-137, 4’-MeO-BDE-17, 6’-MeO-BDE-17, 2’-MeO-BDE=28,
51
5-MeO-BDE-47,
6-MeO-BDE-47,
52
3-MeO-BDE-90, 6-MeO-BDE-90, 3-MeO-BDE-100, 2-MeO-BDE-123, 6-MeO-BDE-137) and
53
glucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP were synthesized by the authors at
54
City University of Hong Kong. Purities of all of the OH-BDEs were greater than 98%.1 All
55
phenolic
C12-BDE-138);
13
C12-BDE-139,
13
C12-BDE-209
C6-2,4-dibromphenol were purchased from Wellington Labs (Ontario, Canada). Three
were
5’-OH-BDE-99,
4’-MeO-BDE-49,
derivatized
by
2’-OH-BDE-68,
6’-OH-BDE-99,
2’-MeO-BDE-68,
reacting
with
6-OH-BDE-85,
3-OH-BDE-100,
6-MeO-BDE-85,
N,O-bis(trimethylsilyl)
3
compounds
2’-OH-BDE-66,
Page
13
13
56
trifluoroacetamide (BSTFA) with 1% trimethylchlorosaline (TMCS) obtained from Acros
57
Organics (Geel, Belgium).
Page
4
58
59
Instrumentation.
60
HPLC-MS/MS
61
Quantification of glucuronide and sulfate conjugates of bromophenols was performed by
62
HPLC–ESI-MS/MS (Agilent 1200 Series HPLC, Agilent Technologies, Waldbronn, Germany)
63
coupled to a MDS Sciex API 3200 QTrap triple quadrupole / linear ion trap mass spectrometer
64
with a Turbo V ion spray source (Applied Biosystems, Foster City, CA, USA). In order to
65
improve sensitivity and selectivity, analytes were detected in Multiple Reaction Monitoring
66
(MRM) mode with a dwell time of 150 ms. The ionization source parameters were as follow: ion
67
spray voltage: -4500kV; curtain gas (N2): 15 psig; collision gas (N2), high; temperature of
68
ionization source, 600oC; ion source gas 1 (nebulizer gas), 60 psig; ion source gas 2 (heater gas),
69
50 pisg. Declustering potential (DP), entrance potential (EP), collision energy (CE) and collision
70
cell exit potential (CXP) of all analytes were optimized to obtain maximum sensitivity. The
71
analytical column was a Waters XBridgeTM C18 2.5 μm 3.0 mm × 50 mm column. A guard
72
column XBridgeTM C18 2.5 μm 3.0 × 20 mm was placed in front of the analytical column.
73
LC separation was accomplished by use of gradient elution at a flow rate of 300 μL/min,
74
with solvent A (5 mM DHAA in Milli-Q) and solvent B (5 mM DHAA in methanol) at the
75
composition of A:B (90:10, v/v) at t = 0 to t = 2 min, changed linearly to A:B (30:70, v/v) over a
76
period of 18 min, then held at such composition for a further 10 min. After the separation, the
77
eluent composition was switched back to A:B (90:10 v/v) and held for 20 min before the next
78
injection. The injection volume was 10 μL.
79
5
GC-NCI-MS
Page
80
81
Identification and quantification of all targeted PBDEs, MeO-BDEs, OH-BDEs and BPs
82
were performed by use of gas chromatography (GC, Agilent 7890A) with
mass-selective
83
detection (MS, Agilent 5975C with triple axis detector) in electron-capture negative ionization
84
(ECNI) mode, by monitoring at m / z = 79 and 81 for most of the congeners, and at m / z = 486.6
85
for BDE-207, BDE-208 and BDE-209. The GC injector was set to 285 oC with an injecting
86
volume of 2 μL. Lesser brominated BDE congeners (mono- to hexa-substituted BDEs),
87
MeO-BDEs, OH-BDEs and BPs were analyzed by use of a 30 m × 0.25 mm × 0.25 μm DB-5MS
88
column, whereas higher BDE congeners (hepta- to deca-substituted BDEs) were analyzed by a
89
15 m × 0.25 mm × 0.1 μm DB-5HT column. The temperature program for the analysis of lesser
90
brominated BDE congeners and MeO-BDEs was as follows: 110 oC for 5 min; 30 oC / min to
91
240 oC; held for 20 min; 30 oC / min to 280 oC held for 2 min and 30 oC / min to 300 oC; held for
92
30 min. The temperature program for the analysis of more brominated BDE congeners was as
93
follows: 60 oC for 5 min; 10 oC / min to 290 oC; held for 2 min; 20 oC / min to 300 oC; held for 9
94
min. The temperature program for the analysis of OH-BDEs and BPs was as follows: 110 oC for
95
5 min; 20 oC / min to 240 oC for 20 min; 30 oC / min to 280 oC; held for 10 min and finally 30 oC
96
/ min to 300 oC; held for 20 min. Total concentrations of different groups of analytes (ΣPBDEs /
97
ΣOH-BDEs / ΣMeO-BDEs / ΣBPs) were reported as the sum of the individual PBDEs /
98
OH-BDEs / MeO-BDEs / BPs congeners quantified.
Quantification
101
Targeted PBDEs, OH-BDEs, MeO-BDEs and BRs in human plasma
102
To avoid photo-degradation, samples were kept in amber vials or sampling tubes wrapped
103
with aluminum foil. Neutral and phenolic fractions of extracts of human blood plasma were
Page
100
6
99
104
slightly modified from previously described methods.2 Each sample of plasma was transferred to
105
a clean glass tube and known amounts of PBDE recovery standards (13C12-BDE-77 and
106
13
107
Hydrochloric acid (2 mL, 6 M) and iso-propanol (6 mL) were added, followed by
108
homogenization. Each sample was extracted by 3 × 6 mL hexane / methyl tert-butyl ether
109
(MTBE). The organic extracts were combined and washed with 1% potassium chloride solution
110
(3 mL). The combined organic extract was evaporated over a gentle steam of nitrogen and lipid
111
content was determined gravimetrically. Lipid was re-dissolved in hexane (4 mL) and partitioned
112
with potassium hydroxide (2 mL, 0.5 M in 50% ethanol) to ionize the phenolic analytes. PBDEs
113
and MeO-BDEs were separated by 3 × 4 mL of hexane. The aqueous layer was acidified by
114
hydrochloric acid (2 mL, 0.5 M), then phenolic compounds were extracted by 3 × 4 mL of
115
hexane / MTBE (9:1, v/v). The neutral and phenolic fractions were blown down to dryness and
116
reconstituted in 5 mL hexane. The neutral fraction was treated with 5 mL of concentrated
117
sulfuric acid twice to remove any lipids.
13
118
The neutral fraction was concentrated over a gentle stream of nitrogen and cleaned-up by
119
passing the concentrate through multilayer column chromatography with 1 g of anhydrous
120
sodium sulfate on top, followed by 8 g of silica and 8 g of alumina. PBDEs and MeO-BDEs were
121
eluted with 50 mL of a hexane / dichloromethane mixture (3:2, v/v). The organic solvent was
122
evaporated to dryness and the sample was reconstituted to 100 μL with 13C12-BDE-139 added as
123
an internal standard for GC / MS analysis.
124
The phenolic fraction was also concentrated under a gentle stream of nitrogen and clean-up
125
by Florisil column chromatography with 1 g of anhydrous sodium sulfate on top of 5 g of Florisil.
126
OH-BDEs and BPs were eluted with 30 mL of a mixture of dichloromethane and hexane (1:1,
7
C12-BDE-209 and 6’-OH-BDE-17 as surrogate standards were added.
Page
C12-BDE-138), and
127
v/v). The organic solvent was evaporated to dryness and the phenolic fraction was derivatized
128
with 100 μL of BSTFA with 1% TMCS at 70 oC for an hour.
129
internal standard for GC / MS analysis.
13
C12-BDE-139 was added as the
130
131
Bromophenol conjugates in human urine
132
The extraction method was similar to that reported in our previous work.1a A human urine
133
sample (5 mL) was partitioned with 3 × 5 mL ethyl acetate. The combined organic solution was
134
evaporated to dryness under a gentle stream of nitrogen. Residues were dissolved in 15 mL of
135
0.67 M sodium acetate buffer at pH 5.2. The resultant solution was applied, at a rate of 1 drop
136
s–1, to an Oasis WAX solid-phase extraction (SPE) cartridge that had been preconditioned
137
sequentially by 5 mL of methanol, 5 mL of Milli-Q water, and 5 mL of 2 M sodium acetate
138
buffer at pH 5.2. The loaded WAX SPE cartridge was then washed in turn by 5 mL of 2 M
139
sodium acetate buffer at pH 5.2, followed by 5 mL of methanol. The glucuronide fraction was
140
then eluted with 4 mL of a formic acid/methanol (1:9, v/v) mixture, and the sulfate fraction was
141
eluted with 4 mL of an aqueous ammonia/methanol (1:9, v/v) mixture. The eluates were
142
evaporated to around 100 μL under a gentle stream of nitrogen. 13C6-2,4-dibromphenol (200 μL,
143
500 ng mL–1) was added as an internal standard for LC-MS/MS quantitation.
144
Quality Assurance and Quality Control.
146
Surrogate standards were used to quantify the concentration of all the BDE congeners using
147
mean relative response factors determined from standard calibration during analysis of human
148
plasma samples. PBDE recovery standards (13C12-BDE-77 and
149
surrogates for mono- to hexa-substituted BDEs and MeO-BDEs, 13C12-BDE-209 was used as the
C12-BDE-138) were used as
8
13
Page
145
150
surrogate for hepta- to deca-substituted BDEs, and 6’-OH-BDE-17 was used as the surrogate for
151
OH-BDEs and BPs. All equipment was rinsed with acetone and hexane to avoid contamination.
152
One laboratory blank and one matrix spike were analyzed for each batch of 18 samples to
153
check for interferences or contamination from solvent and glassware. The method detection limit
154
(MDL) was established by use of lesser concentrations and a consecutive analysis of the series of
155
n spiked samples (Equation 1).
156
MDL = t × σ
157
(1)
158
159
where: σ is the standard deviation of the data and t is the compensation factor from the Student’s
160
t-Table with n – 1 degrees of freedom at a confidence interval of 95%. Method detection limits
161
(MDLs) for BDEs, MeO-BDEs, OH-BDEs and BPs ranged from 0.007 to 0.24 ng/g l.w. For
162
BDE-207, -208, -209, MDLs ranged from 0.15 to 0.75 ng/g l.w. Recoveries of all targeted
163
analytes were within 88 – 103% while the matrix-spiked recoveries were within 78 – 114%.
164
In the analysis of BP conjugates, procedural blanks and matrix spikes were included in each
165
batch of 10 samples. None of the synthesized BPs conjugates were detected in procedural blanks.
166
MDLs of targeted analytes were assessed by use of the same method as that used for plasma:
167
MDLs
168
2,4-dibromophenyl sulfate and 2,4,6-tribromophenyl sulfate were 2.7, 2.9, 2.9 and 2.2 ng/g
169
creatinine, respectively. Recoveries of the analytes were within the range of 72 to 102% and the
170
%RSD ranged from 4 to 9%.
for
2,4-dibromophenyl
glucuronide,
2,4,6-tribromophenyl
glucuronide,
Page
172
9
171
173
310
403
174
175
176
177
178
179
180
181
182
183
184
185
186
*arithmetic mean
187
188
Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various
189
countries.
190
191
194
Page
193
10
192
196
1. (a) Ho, K.L.; Murphy, M.B.; Wan, Y.; Fong, B.M.W.; Tam, S.; Giesy, J.P.; Lam, M.H.W.
197
Anal. Chem. 2012, 84, 9881-9888. (b) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X.; Jones,
198
P.D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J.; Lam, M.H.W.; Giesy, J.P. Environ. Sci.
199
Technol. 2008, 43, 7536-7542.
200
2. (a) Hovander, L.; Athanasiadou, M.; Asplund, L.; Jensen, S.; Klasson-Wehler, E. J. Anal.
201
Toxicol. 2000, 24, 696-703. (b) Qiu, X.; Bigsby, R.M.; Hites, R.A. Environ. Health Prespect.
202
2009, 117, 93-98.
11
References:
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