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Chemosphere 133 (2015) 6–12
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Urinary bromophenol glucuronide and sulfate conjugates: Potentialhuman exposure molecular markers for polybrominated diphenyl ethers
http://dx.doi.org/10.1016/j.chemosphere.2015.03.0030045-6535/� 2015 Elsevier Ltd. All rights reserved.
⇑ Corresponding authors at: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong Special Administrative Reg+852 3442 6888; fax: +852 3442 7406 (M.B. Murphy). Department of Biology & Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong KonAdministrative Region. Tel.: +852 3442 7329; fax: +852 3442 0522 (M.H.-W. Lam).
E-mail addresses: [email protected] (M.B. Murphy), [email protected] (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.
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 Regionb Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Canadac Division of Clinical Biochemistry, Queen Mary Hospital, Hong Kong Special Administrative Regiond Department of Chemistry, Hong Kong Baptist University, Hong Kong Special Administrative Regione Department of Zoology and Center for Integrative Toxicology, Michigan State University, USAf School of Biological Sciences, The University of Hong Kong, Hong Kong Special Administrative Regiong Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USAh State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China
h i g h l i g h t s
� Parallel blood and urine samples werecollected from 100 donors in HongKong.� Levels of selected BP glucuronide and
sulfate conjugates in urine weredetermined.� Their levels were found to correlate
well with that of PBDEs in bloodplasma.� Our results suggest that BP
conjugates can be useful markers forPBDE exposure.
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:Received 10 October 2014Received in revised form 18 February 2015Accepted 1 March 2015Available online 24 March 2015
Handling Editor: Andreas Sjodin
Keywords:BromophenolsPolybrominated diphenyl ethersMetabolitesExposure molecular markers
a b s t r a c t
One possible source of urinary bromophenol (BP) glucuronide and sulfate conjugates in mammalian ani-mal models and humans is polybromodiphenyl ethers (PBDEs), a group of additive flame-retardantsfound ubiquitously in the environment. In order to study the correlation between levels of PBDEs inhuman blood plasma and those of the corresponding BP-conjugates in human urine, concentrations of17 BDE congeners, 22 OH-BDE and 13 MeO-BDE metabolites, and 3 BPs in plasma collected from 100voluntary 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 plasmawere 4.45 ng g�1 lw, 1.88 ng g�1 lw, 0.42 ng g�1 lw and 1.59 ng g�1 lw respectively. Concentrations of glu-curonide and sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP) inpaired samples of urine were determined by liquid chromatography tandem triple quadrupole massspectrometry (LC–MS/MS). BP-conjugates were found in all of the parallel urine samples, in the rangeof 0.08–106.49 lg g�1-creatinine. Correlations among plasma concentrations of RPBDEs/ROH-BDEs/
ion. Tel.:g Special
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 7
Human blood plasmaHuman urine
RMeO-BDEs/RBPs and BP-conjugates in urine were evaluated by multivariate regression and Pearsonproduct correlation analyses. These urinary BP-conjugates were positively correlated with RPBDEs inblood plasma, but were either not or negatively correlated with other organobromine compounds inblood plasma. Stronger correlations (Pearson’s r as great as 0.881) were observed between concentrationsof BDE congeners having the same number and pattern of bromine substitution on their phenyl rings inblood plasma and their corresponding BP-conjugates in urine.
� 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Polybrominated diphenyl ethers (PBDEs), a class of brominatedflame retardants (BFRs), have aroused considerable public concernbecause of their resistance to environmental degradation, espe-cially for lower brominated congeners, their tendency tobioaccumulate 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 per-sistent organic pollutants (POPs) under the Stockholm Convention(Eljarrat and Barceló, 2011). Despite international efforts on therestriction of their production and usage, PBDEs are likely toremain in the global ecosystem for a considerable period of timebecause of their slow rate of degradation for lower brominatedcongeners, and the fact that large amounts of manufactured goodscontaining PBDEs are still in use (Harrad et al., 2006; Betts, 2008).Thus, the continuous monitoring of accumulation of PBDEs inhumans is still important for the accurate assessment of their riskto public health at national and international levels.
The most frequently adopted approach to monitor human expo-sure to PBDEs is the direct quantification of selected BDE congenersand 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 breastmilk (Sudaryanto et al., 2008; Schuhmacher et al., 2009, 2013;Toms et al., 2009; Shi et al., 2013). Other human tissues such ashair, 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 chemi-cal/biochemical analysis and risk assessment is an intrusive opera-tion and difficult to achieve in large-scale population-wide ornational surveys. While sampling of human hair and breast milkcan be considered non-intrusive processes, they face other lim-itations, such as the ease of exogenous contamination of hair sam-ples (Morris et al., 2012; Barbosa et al., 2013) and the gender andage distribution restrictions of sampling breast milk (Landriganet al., 2002). Alternatively, sampling of human urine is simple,quick and non-intrusive, making it much easier to obtain urinesamples from a large number of voluntary donors within a commu-nity for large-scale surveys.
Occurrence of metabolites of selected BDE congeners in urine ofmammalian animal models has already been well established inseveral toxicokinetic studies (Hakk and Letcher, 2003; Chen et al.,2006; Sanders et al., 2006). These metabolites are mainly glu-curonide and sulfate conjugates of dibromophenols (DBPs) and tri-bromophenols (TBPs), probably because of their lower molecularweight (relative to their parent BDE congeners) that facilitatestheir renal removal. Our research team has previously reportedthe synthesis, purification, and characterization of glucuronideand sulfate conjugates of bromophenols (BP) and has developedan analytical protocol for their determination in human urine(Ho et al., 2012). A preliminary survey on 20 voluntary donorsrevealed the presence of at least one of these BP-conjugates intheir urine. In this study, we examined the correlation between
concentrations of PBDEs/OH-BDEs/MeO-BDEs/BPs and in bloodplasma and BP-glucuronide and -sulfate conjugates in urine ofhumans. A total of 100 matched samples of plasma and urine werecollected from volunteer donors in Hong Kong, China. The objectiveof this work was to evaluate whether glucuronide and sulfate con-jugates of BPs in human urine are suitable molecular markers forthe assessment of population exposure to PBDEs.
2. Experimental
2.1. Safety precautions
All necessary precautions were taken during the handling ofsamples of blood and urine. Double latex gloves, facemasks andeye-protection goggles were worn at all times during handling,spiking and transfer of samples from humans. All spent samplesof urine were collected after analysis in separate capped containerswith proper clinical waste labels. Both spent samples and usedpersonal protection items were treated as clinical waste andwere collected and disposed of in accordance with the ‘‘Code ofPractice for the Management of Clinical Waste’’ issued by theEnvironmental Protection Department of the Hong Kong SARGovernment.
2.2. Sample collection
All studies that involved human tissues and body fluids wereconducted in accordance with guidelines of the Research EthicsCommittee of City University of Hong Kong after proper approvalswere given by the Committee. Parallel samples of human plasmaand urine (n = 100; 50 from male and 50 from female donors) werecollected from voluntary donors during March to July 2010 byregistered doctors and nurses at Queen Mary Hospital, HongKong. Besides their gender and age, no other personal informationof those voluntary donors was collected. The age range of thevolunteers was from 16 to 93 years of age (mean ± SD:54.9 ± 21.9 years). These donors were subdivided into differentage 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 phle-botomy technique in vacutainer tubes containing sodium heparinanticoagulant (Vacuette, Greiner bio-one, GmbH, Austria). Wholeblood was then centrifuged at 1500g for 25 min. Plasma wasremoved from the top of the tube. Morning-first urine sampleswere collected in 100 mL sterilized glass bottles and stored at�80 �C within 15 min of sampling until analysis. Urine from eachdonor was subdivided into three replicate samples before low-temperature storage. All samples were carefully labeled anddocumented. Upon analysis, samples were thawed, and 10 mL ofeach sample was retained for creatinine content determination(D’Haese et al., 1985). Creatinine determination was conductedby a kinetic colorimetric assay based on the modified Jaffe methodusing the Roche Modular System (Roche Diagnostics, IN, USA), withan analytical range between 360 and 57500 mmol L�1.
8 K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
2.3. Sample extraction and preparation
Synthesis, purification and characterization of the glucuronideand sulfate conjugates of 2,4-dibromophenol (2,4-DBP) and 2,4,6-tribromophenol (2,4,6-TBP), and the analytical protocols for thequantification of PBDEs, OH-BDEs, MeO-BDEs and BPs in humanplasma and the BP-conjugates in human urine have been describedin 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 givenin 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 Sigmastat3.5 (Sigmastat, Jandel Scientific, CA). Normality of data waschecked by the Klomogorov-Smirnov test. Logarithm, natural-loga-rithm, arcsine, square root, reciprocal square root or cubic roottransformations were used whenever fit to obtain normallydistributed data sets for parametric statistical testing. Student’st-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 datawere not normally distributed, a non-parametric Mann–WhitneyRank Sum test was used for the comparison. One-way ANOVA(parametric) or ANOVA on Ranks (non-parametric) tests were usedto compare concentrations of the target brominated species inplasma and urine among different age categories. Multivariatelinear regression and Pearson product moment correlation analysiswere used to examine the influence of concentrations of PBDEs,MeO-BDEs, OH-BDEs, BPs in blood plasma on concentrations ofBP-conjugates in urine. A P < 0.05 was considered statisticallysignificant for all statistical measurements.
3. Results and discussion
3.1. Concentrations of PBDEs, OMe-PBDEs, OH-PBDEs and BPs inhuman plasma
Concentrations of RPBDEs, RMeO-PBDEs, ROH-PBDEs andRBPs in plasma, normalized by the plasma lipid weight (lw), aresummarized (Table 1). PBDEs were detected in all of the 100human plasma samples, with RPBDEs ranging from 0.01 to18.2 ng g�1 lw. These concentrations of RPBDEs are quitecomparable to those reported in previous studies in other Asiancountries (Tan et al., 2008; Zhu et al., 2009; Uemura et al., 2010;Kim et al., 2012), New Zealand (Harrad and Porter, 2007) and someEuropean countries (Thomas et al., 2006; Gómara et al., 2007;Antignac et al., 2009; Kalantzi et al., 2011), but are lesser thanthose in the population of Northern America (Schecter et al.,2005; Sandanger et al., 2007; Lunder et al., 2010) and those wholive near sites where BFR are produced or used in large quantities(Jin et al., 2009), or e-waste disposal/dismantling areas (Biet al., 2007) (Fig. S1, Supporting Information). Similar to manyprevious studies on the occurrence of PBDEs in human plasma,BDE-47, �99 and �209 were the most abundant BDE congenersdetected in this study, with geometric mean concentrations of0.55 ng g�1 lw; (95% confidence interval: 0.39–0.79 ng g�1 lw),0.33 ng g�1 lw (95% confidence interval: 0.23–0.48 ng g�1 lw), and0.47 ng g�1 lw (95% confidence interval: 0.35–0.62 ng g�1 lw),respectively. There was no significant difference between concen-trations of PBDEs in plasma among all age categories (p = 0.736)or gender (p = 0.143). BDE-47 and �99 were the two most fre-quently detected congeners with a detection frequency of >90%,
while <20% of the plasma samples contained BDE-209. This fre-quency of occurrence of BDE-209 is much less compared to surveyscarried out in the US, European countries and Japan. A recent studyby Wang et al. (2011) reported greater concentrations and occur-rence frequencies of BDE-47 and �99 in seafood from fish marketsin Hong Kong. This suggests that food consumption rather thaninhalation of indoor dust, an environmental matrix with greaterconcentrations of BDE-209, might be a more significant exposureroute to the residents of Hong Kong.
OH-BDEs and BPs were detected in human blood at concentra-tions similar to those of PBDEs. This is consistent with findings ofprevious studies (Athanasiadou et al., 2008; Roosens et al., 2009;Qiu et al., 2009; Wan et al., 2010). 6-OH-BDE-47 and 50-OH-BDE-99 were the two most abundant OH-BDE congeners in the humanblood plasma samples, with geometric mean concentrations of0.32 ng g�1 lw; (95% confidence interval: 0.23–0.45 ng g�1 lw)and 0.06 ng g�1 lw (95% confidence interval: 0.03–0.11 ng g�1 lw),respectively. Three different BPs, namely 2,4-DBP, 2,4,5-tribro-mophenol (2,4,5-TBP) and 2,4,6-TBP, were detected in 80% of theplasma samples. The geometric mean concentration of RBPs was1.59 ng g�1 lw (95% confidence interval: 1.34–1.89 ng g�1 lw). Theoccurrence of these three BPs in mouse plasma after exposure tothe commercial Penta-BDE mixture DE-71 has been reported byQiu et al. (2007). In an in vitro study of the metabolism of BDE-99 by human hepatocytes, Stapleton et al. (2009) also determined2,4,5-TBP in the cell extracts, which was deemed to be generatedby the catabolic cleavage of the diphenyl ether linkage of theBDE congener. These studies suggest that the diphenyl ether cleav-age of PBDEs constitutes one of the sources of BPs (including 2,4-DBP and 2,4,6-TBP) in blood plasma. Other sources may includefood consumption and exposure to other BFRs. 2,4-DBP and2,4,6-TBP have been detected in fresh fish samples commonly con-sumed in Hong Kong (Chung et al., 2003). 2,4,6-TBP has been usedas a flame retardant, and population may be exposed to it in asimilar way as to PBDEs. There was no significant difference inthe concentrations of OH-BDEs and BPs in human plasma betweenmale and female donors (p = 0.749 for OH-BDEs, p = 0.165 for BPs).There were also no significant differences in OH-BDE and BPcontent 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 geo-metric mean concentration of RMeO-BDEs was 0.42 ng g�1 lw (95%confidence interval: 0.31–0.56 ng g�1 lw). This concentration iscomparable to that revealed in a previous study carried out inHong Kong (Wang et al., 2012). 6-MeO-BDE-47 and 4-MeO-BDE-17 were the two most abundant MeO-BDE congeners, with meanconcentrations in human plasma of 0.88 and 0.73 ng g�1 lw,respectively. This pattern of occurrence of MeO-BDEs in humanplasma samples is different from that observed in the US (Qiuet al., 2009), perhaps because some abundant MeO-BDE congeners(6-MeO-BDE-47, 4-MeO-BDE-17, 2-MeO-BDE-28) were notincluded in the previous study. There were no significant differ-ences among concentrations of MeO-BDEs in human plasmaamong all the age groups (p = 0.210), and no statistically significantdifferences in the concentrations of MeO-BDEs were foundbetween male and female donors (p = 0.702).
3.2. Concentrations of BP-glucuronide and -sulfate conjugates inhuman urine samples
To the best of our knowledge, BP-glucuronide and –sulfate con-jugates are not commercially available. Authentic standards of theglucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP forLC–MS/MS determination in this work were obtained via chemicalsynthesis and liquid chromatographic purification. On the otherhand, the presence of conjugates of 2,4,5-TBP in urine samples
Table 1Concentrations (ng g�1 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) 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
RPBDEs 4.45 (3.66–5.41) 0.01–18.20 100 5.13 (4.10–6.41) 0.12–18.20 100 3.82 (2.77–5.28) 0.01–14.18 100RMeO-BDEs 0.42 (0.31–0.56) N.D.–6.87 89 0.44 (0.30–0.65) N.D.–5.15 90 0.40 0.25–0.63 N.D.–6.87 90ROH-BDEs 1.88 (1.53–2.29) N.D.–8.88 83 1.82 (1.40–2.36) N.D.–5.19 86 1.94 (1.43–2.64) N.D.–8.88 80RBPs 1.59 (1.34–1.89) N.D.–7.18 84 1.79 (1.43–2.25) N.D.–5.16 92 1.40 (1.08–3.00) N.D.–7.18 78
N.D.: not detected.a GM: geometric mean.b CI: confidence interval.
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 9
was not determined in this study because of the unavailability of2,4,5-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, withRBP-conjugates ranging from 0.08 to 106.49 lg g�1-creatinine(Table 2). Conjugates of 2,4,6-TBP were the most frequentlydetected, while the frequency of detection of 2,4-DBP conjugateswas around 70%. BP-glucuronides were 5- to 10-fold moreabundant than their corresponding BP-sulfates. There were no sta-tistically significant differences in concentrations of BP-conjugatesin urine among age groups (p = 0.378 for 2,4-DBP-sulfate; p = 0.558for 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 statisticallysignificant difference was observed in the urine concentrationsof 2,4-DBP-glucuronide between male and female donors(p = 0.038), with concentrations of 2,4-DBP-glucuronide found tobe greater in men. A previous study on human urinary bisphenolA (BPA) in Korea also found a similar phenomenon where concen-trations of BPA-glucuronides in men were greater than those inwomen.
3.3. Correlations between BP-glucuronide and -sulfate conjugates andRPBDEs, ROMe-PBDEs, ROH-PBDEs and RBPs
Multivariate linear regression analysis was employed to explorethe relationship among different forms of BDEs/BPs in bloodplasma and BP-conjugates in urine. The standardized regressioncoefficient b was used to quantify the relationship between BP-conjugates and the various determinants. The first regressionmodel was built using the natural log-transformed summation ofconcentrations of PBDEs, OH-BDEs, MeO-BDEs and BPs, i.e.lnRPBDEs, lnROH-PBDEs, lnRMeO-PBDEs and lnRBPs, in plasmasamples as independent variables, and the sum of all four BP-glucuronide and -sulfate conjugates, i.e. lnRBP-conjugates, in urinesamples as the dependent variable (Table 3). The coefficient ofdetermination (R2) of the regression model established wasonly 0.223, and the model was significantly affected by bothlnRPBDEs and lnRBPs, with their standardized regression coeffi-cients (b) being 0.716 and �0.585 respectively (p < 0.001 forlnRPBDEs and p = 0.013 for lnRBPs).
While there might not be simple relation between total concen-trations of PBDEs in human blood and that of total BP-conjugates inhuman urine, we explored correlations among structurally-relatedorganobromine species in blood and in urine. Based on numerousprevious in vivo studies on laboratory mice and in vitro studieson human cells, catabolic cleavage of PBDEs at the ether linkagecan produce water-soluble glucuronide and sulfate BP-conjugateswith the number and relative position of the bromine-substituentsresembling 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 conju-gates of 2,4-DBP/2,4,6-TBP in urine were investigated (Tables 4aand 4b). The R2 values increased to 0.744 (lnR2,4-DBP-conjugates)and 0.707 (lnR2,4,6-TBP-conjugates), which suggests that therefined regression models better explained the variability of thedependents. They also gave better standardized coefficients (b):4.34 for lnR2,4-dibromo-BDEs and 3.23 for lnR2,4,6-tribromo-BDEs, revealing much stronger relationships between R2,4-di-bromo-BDEs/R2,4,6-tribromo-BDEs in human plasma and theircorresponding BP-conjugates in urine. All the variance inflationfactors (VIFs) of the independent variables were <2, indicatingthe absence of multicollinearity in the regression models. Thus,our results revealed strong relationships between BP-glucuronideand -sulfate conjugates in human urine and R2,4-dibromo-BDEsand R2,4,6-tribromo-BDEs in human blood plasma. On the otherhand, their relationships with RMeO-BDEs, ROH-BDEs, or RBPsin blood plasma were of less significance. These results suggestthat urinary BP-conjugates in human originated from exposure toPBDEs rather than MeO-BDEs, OH-BDEs or BPs.
Pearson product moment correlation was used to evaluate thecorrelations between natural-log-transformed RBP-conjugatesand RPBDEs, RMeO-BDEs, ROH-BDEs and RBPs. Strong relation-ships were observed between lnR2,4-dibromo-BDEs in humanblood vs lnR2,4-DBP-conjugates in human urine and lnR2,4,6-tri-bromo-BDEs in human blood vs lnR2,4,6-TBP-conjugates in humanurine (Pearson’s r = 0.881 and 0.823, respectively; Tables 5a and5b). These results demonstrate the correlation between urinaryconcentration of BP-glucuronide and -sulfate conjugates and thelevel of PBDEs in blood plasma (Figs. 1 and 2). On the other hand,concentrations of urinary BP-glucuronide and -sulfate conjugatesdo not correlate well with those of lnRMeO-BDEs, lnROH-BDEsand lnRBPs in blood plasma. This suggests that the glucuronideand sulfate conjugates of BPs in human urine may be useful asmolecular markers for human exposure to PBDEs. It is arguablethat these BP-conjugates may only be able to reflect human expo-sure to PentaBDEs, but not OctaBDEs and DecaBDE. The apparenthalf-life of OctaBDEs and DecaBDE in human serum are less than91 days (Thuresson et al., 2006), which is much shorter than thatof 2 years for PentaBDEs (Geyer et al., 2004). Thus, even thoughthe 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 betweenR2,4,5-tribromo-BDEs in human blood and R2,4,5-TBP-conjugatesin human urine was not explored in this study. Also, the presenceof BDE-glucuronide and -sulfate conjugates in human urine wasnot determined because of the unsufficient quantity of thecorresponding OH-BDEs available for the chemical synthesis ofthe metabolites. Their aptness as molecular markers for populationexposure to PBDEs will have to be addressed in the future. Infantsand children were not included in this study because of ethicalconsiderations associated with the collection of their blood and
Table 2Concentrations (lg g�1 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) 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
2,4-DBP glucuronide 0.32 (0.23–0.44) N.D.–23.81 71 0.42 0.27–0.64 0.01–23.81 76 0.21 (0.13–0.34) N.D.–7.52 662,4-DBP sulfate 0.11 (0.08–0.14) N.D.–2.08 86 0.10 (0.07–0.15) N.D.–2.08 88 0.11 (0.08–0.17) N.D.–2.08 842,4,6-TBP glucuronide 0.87 (0.58–1.30) N.D.–102.21 68 0.98 (0.51–1.73) N.D.–102.21 54 0.80 (0.47–1.38) N.D.–44.08 822,4,6-TBP sulfate 0.10 (0.08–0.13) N.D.–2.93 94 0.10 (0.07–0.15) N.D.–2.93 94 0.10 (0.07–0.14) N.D.–2.93 94
N.D.: not detected.a GM: geometric mean.b CI: confidence interval.
Table 3Significant independent variables of urinary concentrations of BP-conjugates revealedby multivariate linear regression analysis.
Model summaryR2 0.223
Independent variable ba Pb VIFc
lnRPBDEs 0.716 <0.001 1.056lnRMeO-BDEs 0.063 0.632 1.059lnROH-BDEs �0.23 0.239 1.041lnRBPs �0.585 0.013 1.056
Dependent value was urinary lnR[BP-conjugates].a 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.
Table 4bSignificant independent variables of urinary concentrations of 2,4,6-TBP-conjugatesrevealed by multivariate linear regression analysis.
Model summaryR2 0.707
Independent variable ba Pb VIFc
lnR2,4,6-Tribromo-BDEs 3.226 <0.001 1.005lnR2,4,6-TBP 0.135 0.428 1005
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.
Table 5aCorrelation 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 P r P r P
lnR2,4-Dibromo-BDEs 0.881 <0.05 0.871 <0.05 0.884 <0.05lnR2,4-Dibromo-MeO-BDEs �0.080 0.449 �0.047 0.77 �0.100 0.526lnR2,4-Dibromo-OH-BDEs �0.095 0.452 0.053 0.743 �0.200 0.221lnR2,4-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 5bCorrelation coefficients between urinary 2,4,6-TBP-conjugates and the variouscongeners PBDEs and BPs in human plasma.
10 K.-L. Ho et al. / Chemosphere 133 (2015) 6–12
urine samples. However, previous studies have revealed aninverted age-dependent accumulation of PBDEs, perhaps becauseof the dietary preferences, greater frequency of hand-to-monthactivities 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 betweenplasma PBDEs and urinary BP-conjugates in children. Another areathat needs further study is potential ethnic differences in the effi-cacy of PBDE metabolism. Previous studies have shown thatgreater glucuronidation of morphine occurred in Chinese peoplecompared to Caucasians (Zhou et al., 1993). Alternatively, ethnicChinese were less able to metabolize codeine by glucuronidation(Yue et al., 1989, 1991). Results from Goldzieher and coworkerhave shown that the pattern of glucuronide conjugation as wellas oxidative metabolism of estrogens (ethinyl estradiol) differedamong Nigerian, Sri Lankan and American populations (Williamsand Goldzieher, 1980; Goldzieher and Brody, 1990). Another studyof the excretion of N-glucuronide conjugates of nicotine and
Table 4aSignificant independent variables of urinary concentrations of 2,4-DBP-conjugatesrevealed by multivariate linear regression analysis.
Model summaryR2 0.744
Independent variable ba Pb VIFc
lnR2,4-Dibromo-BDEs 4.34 <0.001 1.089lnR2,4-Dibromo-MeO-BDEs 0.0138 0.834 1.071lnR2,4-Dibromo-OH-BDEs 0.0682 0.547 1.001lnR2,4-DBP 0.0345 0.617 1.059
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.
Total (n = 100) Male (n = 50) Female (n = 50)
r P r P r P
lnR2,4,6-Tribromo-BDEs 0.823 <0.05 0.820 <0.05 0.834 <0.05lnR2,4,6-TBP �0.007 0.907 �0.06 0.699 �0.074 0.655
Analysis of urinary BP conjugates were conducted after natural-log transformation.
cotinine has shown that people with African origins excreted sig-nificantly less glucuronide conjugates than Caucasians (Caraballoet al., 1998; Benowitz et al., 1999). Information on the differencesin the metabolism of POPs among ethnic groups is scarce. Mattersare further complicated by the differences in the compositions ofPBDE mixtures used in different parts of the world, and dietaryhabits of people. Zota and coworkers (Zota et al., 2008) have shownthat PBDE exposure at different regions of the US was different.
Fig. 1. Correlation analyses between the concentration of natural logarithm-transformed R2,4-dibromo-BDEs in human plasma and natural logarithm-transformed urinary R2,4-DBP-conjugates.
Fig. 2. Correlation analyses between the concentration of natural logarithm-transformed R2,4,6-tribromo-BDEs in human plasma and natural logarithm-transformed urinary R2,4,6-TBP-conjugates.
K.-L. Ho et al. / Chemosphere 133 (2015) 6–12 11
Acknowledgements
This work is support by a grant from Research Grants Council ofthe 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, theP.R. China to Nanjing University and the Einstein ProfessorProgram of the Chinese Academy of Sciences. He was also sup-ported by the Canada Research Chair program, a VisitingDistinguished Professorship in the Department of Biology andChemistry and State Key Laboratory in Marine Pollution, CityUniversity of Hong Kong.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2015.03.003.
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Urinary Bromophenol Glucuronide and Sulfate Conjugates: Potential Human 2
Exposure Molecular Markers for Polybrominated Diphenyl Ethers 3
4
Ka-Lok Ho,a Man-Shan Yau. a Margaret B. Murphy, *a Yi Wan,†b Bonnie M. –W. Fong,c,d Sidney Tam,c 5
John P. Giesy,a,b,,e,f,g,h Kelvin S. –Y. Leung,d Michael H. –W. Lam*a 6
7
8
9
10
Supporting Information 11
12
13
14
15
16
17
18
19
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Table of Content 21
Methods 22
Materials and General Procedures 23
Instrumentation 24
Identification and Quantification 25
Quality Assurance and Quality Control 26
27
Figures 28
Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various 29
countries. 30
31
References 32
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3
Methods 33
Materials and General Procedures. 34
All starting materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as 35
received unless stated otherwise. Oasis WAX® (6 mL / 150 mg) and HLB® (6 mL / 200 mg) 36
cartridges were obtained from Waters Corp. (Milford, MA, USA). Dihexylammonium acetate 37
(DHAA) was obtained from Sigma-Aldrich. Standards for polybrominated diphenylethers 38
(PBDE), including BDE-3, BDE-15, BDE-28, BDE-47, BDE-66, BDE-85, BDE-99, BDE-100, 39
BDE-153, BDE-154, BDE-183, BDE-184, BDE-197, BDE-202, BDE-207, BDE-208, BDE-209; 40
recovery spike standard (13C12-BDE-77 and 13C12-BDE-138); 13C12-BDE-139, 13C12-BDE-209 41
and 13C6-2,4-dibromphenol were purchased from Wellington Labs (Ontario, Canada). Three 42
standards for bromophenols (BP), 2,4-dibromophenol (2,4-DBP), 2,4,5-tribromophenol 43
(2,4,5-TBP) and 2,4,6-tribromophenol (2,4,6-TBP), and five hydroxylated-PBDE (OH-BDE) 44
(4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 5’-OH-BDE-99 and 6’-OH-BDE-99) standards 45
were purchased from Accustandard (New Haven, Connecticut, USA). All other OH-BDEs, 46
MeO-BDEs (2’-OH-BDE-7, 3’-OH-BDE-7, 4’-OH-BDE-17, 6’-OH-BDE-17, 2’-OH-BDE-28, 47
6-OH-BDE-47, 4’-OH-BDE-49, 2’-OH-BDE-66, 2’-OH-BDE-68, 6-OH-BDE-85, 48
4-OH-BDE-90, 6-OH-BDE-90, 5’-OH-BDE-99, 6’-OH-BDE-99, 3-OH-BDE-100, 49
2-OH-BDE-123, 6-OH-BDE-137, 4’-MeO-BDE-17, 6’-MeO-BDE-17, 2’-MeO-BDE=28, 50
5-MeO-BDE-47, 6-MeO-BDE-47, 4’-MeO-BDE-49, 2’-MeO-BDE-68, 6-MeO-BDE-85, 51
3-MeO-BDE-90, 6-MeO-BDE-90, 3-MeO-BDE-100, 2-MeO-BDE-123, 6-MeO-BDE-137) and 52
glucuronide and sulfate conjugates of 2,4-DBP and 2,4,6-TBP were synthesized by the authors at 53
City University of Hong Kong. Purities of all of the OH-BDEs were greater than 98%.1 All 54
phenolic compounds were derivatized by reacting with N,O-bis(trimethylsilyl) 55
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trifluoroacetamide (BSTFA) with 1% trimethylchlorosaline (TMCS) obtained from Acros 56
Organics (Geel, Belgium). 57
58
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Instrumentation. 59
HPLC-MS/MS 60
Quantification of glucuronide and sulfate conjugates of bromophenols was performed by 61
HPLC–ESI-MS/MS (Agilent 1200 Series HPLC, Agilent Technologies, Waldbronn, Germany) 62
coupled to a MDS Sciex API 3200 QTrap triple quadrupole / linear ion trap mass spectrometer 63
with a Turbo V ion spray source (Applied Biosystems, Foster City, CA, USA). In order to 64
improve sensitivity and selectivity, analytes were detected in Multiple Reaction Monitoring 65
(MRM) mode with a dwell time of 150 ms. The ionization source parameters were as follow: ion 66
spray voltage: -4500kV; curtain gas (N2): 15 psig; collision gas (N2), high; temperature of 67
ionization source, 600oC; ion source gas 1 (nebulizer gas), 60 psig; ion source gas 2 (heater gas), 68
50 pisg. Declustering potential (DP), entrance potential (EP), collision energy (CE) and collision 69
cell exit potential (CXP) of all analytes were optimized to obtain maximum sensitivity. The 70
analytical column was a Waters XBridgeTM C18 2.5 μm 3.0 mm × 50 mm column. A guard 71
column XBridgeTM C18 2.5 μm 3.0 × 20 mm was placed in front of the analytical column. 72
LC separation was accomplished by use of gradient elution at a flow rate of 300 μL/min, 73
with solvent A (5 mM DHAA in Milli-Q) and solvent B (5 mM DHAA in methanol) at the 74
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 75
period of 18 min, then held at such composition for a further 10 min. After the separation, the 76
eluent composition was switched back to A:B (90:10 v/v) and held for 20 min before the next 77
injection. The injection volume was 10 μL. 78
79
GC-NCI-MS 80
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Identification and quantification of all targeted PBDEs, MeO-BDEs, OH-BDEs and BPs 81
were performed by use of gas chromatography (GC, Agilent 7890A) with mass-selective 82
detection (MS, Agilent 5975C with triple axis detector) in electron-capture negative ionization 83
(ECNI) mode, by monitoring at m / z = 79 and 81 for most of the congeners, and at m / z = 486.6 84
for BDE-207, BDE-208 and BDE-209. The GC injector was set to 285 oC with an injecting 85
volume of 2 μL. Lesser brominated BDE congeners (mono- to hexa-substituted BDEs), 86
MeO-BDEs, OH-BDEs and BPs were analyzed by use of a 30 m × 0.25 mm × 0.25 μm DB-5MS 87
column, whereas higher BDE congeners (hepta- to deca-substituted BDEs) were analyzed by a 88
15 m × 0.25 mm × 0.1 μm DB-5HT column. The temperature program for the analysis of lesser 89
brominated BDE congeners and MeO-BDEs was as follows: 110 oC for 5 min; 30 oC / min to 90
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 91
30 min. The temperature program for the analysis of more brominated BDE congeners was as 92
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 93
min. The temperature program for the analysis of OH-BDEs and BPs was as follows: 110 oC for 94
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 95
/ min to 300 oC; held for 20 min. Total concentrations of different groups of analytes (ΣPBDEs / 96
ΣOH-BDEs / ΣMeO-BDEs / ΣBPs) were reported as the sum of the individual PBDEs / 97
OH-BDEs / MeO-BDEs / BPs congeners quantified. 98
99
Quantification 100
Targeted PBDEs, OH-BDEs, MeO-BDEs and BRs in human plasma 101
To avoid photo-degradation, samples were kept in amber vials or sampling tubes wrapped 102
with aluminum foil. Neutral and phenolic fractions of extracts of human blood plasma were 103
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slightly modified from previously described methods.2 Each sample of plasma was transferred to 104
a clean glass tube and known amounts of PBDE recovery standards (13C12-BDE-77 and 105
13C12-BDE-138), and 13C12-BDE-209 and 6’-OH-BDE-17 as surrogate standards were added. 106
Hydrochloric acid (2 mL, 6 M) and iso-propanol (6 mL) were added, followed by 107
homogenization. Each sample was extracted by 3 × 6 mL hexane / methyl tert-butyl ether 108
(MTBE). The organic extracts were combined and washed with 1% potassium chloride solution 109
(3 mL). The combined organic extract was evaporated over a gentle steam of nitrogen and lipid 110
content was determined gravimetrically. Lipid was re-dissolved in hexane (4 mL) and partitioned 111
with potassium hydroxide (2 mL, 0.5 M in 50% ethanol) to ionize the phenolic analytes. PBDEs 112
and MeO-BDEs were separated by 3 × 4 mL of hexane. The aqueous layer was acidified by 113
hydrochloric acid (2 mL, 0.5 M), then phenolic compounds were extracted by 3 × 4 mL of 114
hexane / MTBE (9:1, v/v). The neutral and phenolic fractions were blown down to dryness and 115
reconstituted in 5 mL hexane. The neutral fraction was treated with 5 mL of concentrated 116
sulfuric acid twice to remove any lipids. 117
The neutral fraction was concentrated over a gentle stream of nitrogen and cleaned-up by 118
passing the concentrate through multilayer column chromatography with 1 g of anhydrous 119
sodium sulfate on top, followed by 8 g of silica and 8 g of alumina. PBDEs and MeO-BDEs were 120
eluted with 50 mL of a hexane / dichloromethane mixture (3:2, v/v). The organic solvent was 121
evaporated to dryness and the sample was reconstituted to 100 μL with 13C12-BDE-139 added as 122
an internal standard for GC / MS analysis. 123
The phenolic fraction was also concentrated under a gentle stream of nitrogen and clean-up 124
by Florisil column chromatography with 1 g of anhydrous sodium sulfate on top of 5 g of Florisil. 125
OH-BDEs and BPs were eluted with 30 mL of a mixture of dichloromethane and hexane (1:1, 126
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v/v). The organic solvent was evaporated to dryness and the phenolic fraction was derivatized 127
with 100 μL of BSTFA with 1% TMCS at 70 oC for an hour. 13C12-BDE-139 was added as the 128
internal standard for GC / MS analysis. 129
130
Bromophenol conjugates in human urine 131
The extraction method was similar to that reported in our previous work.1a A human urine 132
sample (5 mL) was partitioned with 3 × 5 mL ethyl acetate. The combined organic solution was 133
evaporated to dryness under a gentle stream of nitrogen. Residues were dissolved in 15 mL of 134
0.67 M sodium acetate buffer at pH 5.2. The resultant solution was applied, at a rate of 1 drop 135
s–1, to an Oasis WAX solid-phase extraction (SPE) cartridge that had been preconditioned 136
sequentially by 5 mL of methanol, 5 mL of Milli-Q water, and 5 mL of 2 M sodium acetate 137
buffer at pH 5.2. The loaded WAX SPE cartridge was then washed in turn by 5 mL of 2 M 138
sodium acetate buffer at pH 5.2, followed by 5 mL of methanol. The glucuronide fraction was 139
then eluted with 4 mL of a formic acid/methanol (1:9, v/v) mixture, and the sulfate fraction was 140
eluted with 4 mL of an aqueous ammonia/methanol (1:9, v/v) mixture. The eluates were 141
evaporated to around 100 μL under a gentle stream of nitrogen. 13C6-2,4-dibromphenol (200 μL, 142
500 ng mL–1) was added as an internal standard for LC-MS/MS quantitation. 143
144
Quality Assurance and Quality Control. 145
Surrogate standards were used to quantify the concentration of all the BDE congeners using 146
mean relative response factors determined from standard calibration during analysis of human 147
plasma samples. PBDE recovery standards (13C12-BDE-77 and 13C12-BDE-138) were used as 148
surrogates for mono- to hexa-substituted BDEs and MeO-BDEs, 13C12-BDE-209 was used as the 149
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surrogate for hepta- to deca-substituted BDEs, and 6’-OH-BDE-17 was used as the surrogate for 150
OH-BDEs and BPs. All equipment was rinsed with acetone and hexane to avoid contamination. 151
One laboratory blank and one matrix spike were analyzed for each batch of 18 samples to 152
check for interferences or contamination from solvent and glassware. The method detection limit 153
(MDL) was established by use of lesser concentrations and a consecutive analysis of the series of 154
n spiked samples (Equation 1). 155
156
MDL = t × σ (1) 157
158
where: σ is the standard deviation of the data and t is the compensation factor from the Student’s 159
t-Table with n – 1 degrees of freedom at a confidence interval of 95%. Method detection limits 160
(MDLs) for BDEs, MeO-BDEs, OH-BDEs and BPs ranged from 0.007 to 0.24 ng/g l.w. For 161
BDE-207, -208, -209, MDLs ranged from 0.15 to 0.75 ng/g l.w. Recoveries of all targeted 162
analytes were within 88 – 103% while the matrix-spiked recoveries were within 78 – 114%. 163
In the analysis of BP conjugates, procedural blanks and matrix spikes were included in each 164
batch of 10 samples. None of the synthesized BPs conjugates were detected in procedural blanks. 165
MDLs of targeted analytes were assessed by use of the same method as that used for plasma: 166
MDLs for 2,4-dibromophenyl glucuronide, 2,4,6-tribromophenyl glucuronide, 167
2,4-dibromophenyl sulfate and 2,4,6-tribromophenyl sulfate were 2.7, 2.9, 2.9 and 2.2 ng/g 168
creatinine, respectively. Recoveries of the analytes were within the range of 72 to 102% and the 169
%RSD ranged from 4 to 9%. 170
171
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174
175
176
177
178
179
180
181
182
183
184
185
186
187
Figure S1. Median concentrations of PBDEs in blood plasma of human blood in various 188
countries. 189
190
191
192
193
194
403 310
*arithmetic mean
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References: 195
1. (a) Ho, K.L.; Murphy, M.B.; Wan, Y.; Fong, B.M.W.; Tam, S.; Giesy, J.P.; Lam, M.H.W. 196
Anal. Chem. 2012, 84, 9881-9888. (b) Wan, Y.; Wiseman, S.; Chang, H.; Zhang, X.; Jones, 197
P.D.; Hecker, M.; Kannan, K.; Tanabe, S.; Hu, J.; Lam, M.H.W.; Giesy, J.P. Environ. Sci. 198
Technol. 2008, 43, 7536-7542. 199
2. (a) Hovander, L.; Athanasiadou, M.; Asplund, L.; Jensen, S.; Klasson-Wehler, E. J. Anal. 200
Toxicol. 2000, 24, 696-703. (b) Qiu, X.; Bigsby, R.M.; Hites, R.A. Environ. Health Prespect. 201
2009, 117, 93-98. 202