Urinary bromophenols phase II conjugates: A novel human exposure biomarker for PBDEs
Ka-Lok Ho1, Bonnie Mei-Wah Fong2,3, Yi Wan4, Sidney Tam3,
John P. Giesy1,4,5, Kelvin Sze-Yin Leung2, Michael Hon-Wah Lam1*
Key Laboratory for Marine Pollution, Department of Biology & Chemistry, City University of Hong Kong, Hong Kong SAR,
2Department of Chemistry, Hong Kong Baptist University, Hong Kong SAR,
3Department of Clinical Biochemistry, Queen Mary Hospital, Hong Kong SAR,
4Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada,
5Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
1. Introduction
Human adipose tissues and blood are the most frequently analyzed
samples for the estimation of population exposure to POPs e.g. PBDEs.
However, sampling of these tissues are intrusive operations and it is usually
difficult to carry out large-scale survey. Sampling of human breast milk can
be considered a non-intrusive process, but samples are only restricted to
lactating women of a narrow range of ages. It may not be able to truly
reflect the level of contaminant exposure to the population. Thus, We aim
to develop non-intrusive, representative and convenient exposure markers
for selected environmental contaminants. Sampling of human urine is a
truly non-intrusive process and samples are much easier to obtain from
voluntary donors, for large-scale exposure surveys. It would be ideal if the
presence of PBDE metabolites / PBDE congeners in human urine can be
used as biomarkers for human exposure to PBDEs. Pharmacokinetic and
toxicokinetic studies of BDE-47 and BDE-99 in mammalian models have
already identified the presence of Phase II metabolites, mainly
dibromophenols (DBPs), tribromophenol (TBPs) glucuronide and sulfate
conjugates, in urine samples.
Table 2a
Table 2b
Human plasma samples (n = 100)
Min – Max
% of Detection
0.01 – 23.47
100
(ng / g l.w. )
6.38 ± 4.72
ΣBDEs
Human urine samples (n = 100)
Mean ± S.D.
Mean ± S.D.
ΣMeO-BDEs
0.94 ± 1.39
N.D. – 6.87
89
ΣOH-BDEs
2.69 ± 2.16
N.D. – 8.88
82
ΣBRPs
1.90 ± 1.84
N.D. – 7.18
84
% of
Min – Max
(µg / g creatinine)
Detection
2,4-DBPG
1.09 ± 3.15
N.D. – 23.81
71
2,4- DBPS
0.21 ± 0.34
N.D. – 2.08
86
2,4,6- TBPG
5.16 ±14.76
N.D. – 102.21
68
2,4,6- TBPS
0.22 ± 0.42
N.D. – 2.93
94
Table 2a. Mean Concentration of ΣPBDE, ΣMeO-BDEs, ΣOH-BDEs & ΣBromophenols in human plasma
Table 2b. Mean Concentration of bromophenols glucuronide and sulfate conjugates in human urine
3. Linear Regression & Correlation
 Σln[BP-glucuronide] and Σln[BP-sulfate] in
human urine increases with increasing
concentration of ΣPBDEs in the human plasma
20
Y = 5.616 + 1.2939x
18
2
r = 0.2582
16
ΣPBDEs (ng/g lipid Wieght)
1State
14
12
 ΣPBDEs & ΣBRPs are statistically significant in
accounting for the presence of bromophenols
glucuronide and sulfate conjugates in human
urine
10
8
6
4
2
 Pearson product moment correlation shows a
positive, moderate correlation between
ΣPBDEs in human plasma and Σ[BPglucuronide] and Σ[BP-sulfate] in human urine
while the other determinants show negatively
correlated
0
-2
-3
-2
-1
0
1
2
3
4
5
ΣlnBromophenol glucuronide and sulfate conjugates
Fig. 3a. Scattered plot of ΣBP conjugates in human urine against
ΣPBDEs in human plasma
2. Sample Collection & Analytical Methods
Parallel blood plasma and urine samples from 100 adult voluntary
donors (50 males, 50 females) have been collected.
Extraction method of individual PBDE, OH-BDE, MeO-BDEs, and
bromophenols in human plasma was followed the published method
with slightly modifications
Solid phase extraction (Oasis® WAX) was developed in house and
adopted to extract bromophenols glucuronide and sulfate conjugates in
human urine.
Multiple Linear Regression
MeO-BDEs, OH-BDEs, BRPs were identified in human plasma samples
with lower concentrations compared to their parent PBDEs.
Bromophenol glucuronide and sulfate conjugates were quantified in
human urine samples.
Moment
 To further explore the correlation between
PBDEs in the blood plasma and bromophenol
β
P
VIF
r
P
metabolites in the urine, multivariant linear
ΣPBDEs
3.568 <0.001 1.132
0.508 <0.001
regression models for those Phase II
ΣMeO-BDEs
1.283
0.204 1.146 -0.243 0.0211
metabolites with their corresponding parent
ΣOH-BDEs
0.284
0.777 1.485 -0.0981 0.378
BDE congeners: i.e. BDEs, MeO-BDEs, OH-BDEs
ΣBRPs
2.952
0.004 1.364 -0.329 0.002
and bromophenols with 2,4-dibromo- and
Table 3a. Significant predictors of the urinary concentration of bromophenol
2,4,6-tribromo substitution, were examined.
conjugates (natural log transformed)
Adjusted R2
0.358
 With eliminating some influences cases, the
goodness of fits (R2) are improved a lot in both
models.
3. PBDE Concentrations
The population exposure of PBDEs in Hong Kong was found to be
comparable to that in other major cities in Asia and Europe, but lower
than that in the Northern America and other industrialized regions.
Pearson Product
lnΣ2,4-dibromophenol conjugates
Pearson Product
Multiple Linear Regression
Adjusted R2
Moment
 PBDEs are taken a great part into account the
presence of bromophenol glucuronide and
sulfate conjugate in urine
0.828
β
P
Model summary for lnΣ2,4,6-tribromophenol conjugates
VIF
r
P
Pearson Product
Multiple Linear Regression
Σ2,4-Dibromo-BDEs
13.599
<0.001
1.128
0.898
Adjusted R2
Σ2,4-Dibromo-
2.316
0.025
1.172
-0.278
Moment
<0.001
0.749
β
P
VIF
r
P
Σ2,4,6-Tribromo-BDEs
12.330
<0.001
1.018
0.863
<0.001
Σ2,4,6-TBP
1.196
0.237
1.018 -0.105
0.346
0.001
MeO-BDEs
Σ2,4-Dibromo-OH-
1.907
0.062
1.30
-0.0926
0.414
BDEs
Σ2,4-DBP
0.785
0.436
1.173
-0.154
0.195
Table 3c. Significant predictors of the urinary concentration of 2,4,6tribromophenol conjugates (natural log transformed data)
Table 3b. Significant predictors of the urinary concentration of 2,4dibromophenol conjugates (natural log transformed data)
Y = 1.2939 + 0.1891x
1.8
Σ2,4,6-Tribromo-BDEs (ng/g lipid Wieght)
Σ2,4-Dibromo-BDEs (ng/g lipid Wieght)
2.0
2
r = 0.8066
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
-6
-4
-2
0
2
4
Y = 0.147 + 0.025x
0.25
2
r = 0.7455
0.20
0.15
0.10
0.05
0.00
-4
-2
0
2
4
6
Σln2,4,6-Tribromophenol glucuronide and sulfate conjugates
Σln2,4-Dibromophenol glucuronide and sulfate conjugates
Fig. 3b. Scattered plots of ΣBP-conjugates in human urine vs Σ(structural analog)BDEs n blood plasma:
(Left) Σ2,4-dibromophenol conjugates vs Σ2,4-dibromo-BDEs & (Right) Σ2,4,6-tribromophenol conjugates vs Σ2,4,6-tribromo-BDEs
4. Summary
To the best of our knowledge, this is the first study to show a significant relationship
between plasma PBDEs and urinary bromophenols conjugates in human. This result is
important because it confirms that BRP-conjugates in human urine are derived from
PBDEs but not bromophenols and the controversial BDE-metabolites, such as OMeBDEs and OH-BDEs. High correlation factors indicate that these urinary bromophenols
conjugates are promising biomarkers for the assessment of population PBDE exposure.
5. Acknowledgement:
Fig. 2a. International comparisons of BDE congeners in human blood sample
This work is supported by a grant from Research Grants Council of the Hong Kong
Special Administrative Region, China [Reference No. CityU 9041623]