Urinary bisphenol A detection is significantly associated with young and obese Thai children
Article Category: Brief communication (Original)
Published Online: Jan 31, 2017
Page range: 363 - 372
DOI: https://doi.org/10.5372/1905-7415.0903.405
Keywords
© 2015 Sopon Pornkunwilai, Wichit Nosoongnoen, Chutima Jantarat, Suttipong Wachrasindhu, Vichit Supornsilchai
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
Bisphenol A (BPA), a xenoestrogenic monomer, is a common industrial chemical and is used to synthesize polycarbonate plastic polymers, which are applied to manufacture of various products such as compact discs, automobile parts, plastic water and baby bottles, plastic dinnerware, eyeglass lenses, toys, and thermal paper, and epoxy resin linings in cans for food products [1]. Over 3 thousand million kilograms of BPA are produced each year. Recent studies have shown that dietary ingestion is a major route (99%) of human exposure to BPA contamination, especially from canned foods [2, 3]. Other routes are microwave containers, soft drinks, smoking, alcohol consumption, dental sealants [4], medical procedures [5], and plastic tubing [6]. BPA was detected in about 93%-100% of children and adolescents in Northern America, some European nations, Egypt, Australia, and Asian countries [7-17]. The only report from Thailand found that BPA was detected 52.8% in serum samples in adults [18]. Negative health outcomes have been associated with bisphenol A. BPA is postulated to alter pancreatic β-cell functions [19, 20] and thyroid hormone pathways [18]. There have been links with insulin resistance [20-21], type 2 diabetes mellitus [6, 22, 23], obesity [24-30], coronary heart disease [31], coronary artery stenosis [32], precocious puberty in girls [33], and behavioral problems including anxiety, depression, and hyperactivity [34, 35]. Obesity is an increasing health problem in Thailand. Obesity is related with comorbidities, such as metabolic and cardiovascular diseases [36]. The prevalence of obesity in Thai children is 7.9%-10.8% and the trend has been increasing [37-39]. Obesity is a complex, multifactorial disease and its etiology involves the interaction between both genetic and environmental factors including endocrine disruptors (EDs). An association between urinary BPA concentrations and obesity has previously been reported [24-30]. To our knowledge, the present study is the first study of BPA exposure in Thai children and adolescents using the standardized and highly sensitive technique of liquid chromatography tandem mass spectrometry (LC-MSMS) to determine BPA in urine. Urine is an appropriate specimen with which to determine BPA exposure because BPA and its metabolites are present in urine for several weeks or months [40, 41]. BPA is rapidly eliminated from plasma within 24 hours after ingestion [41].
This cross-sectional study was conducted between September 2012 and January 2013. Participants were recruited from two schools, one kindergarten-elementary school and one middle school in the Patumwan District of Bangkok. Consent forms were signed by the parents of the 482 students we attempted to recruit for the study. Ninety-two students were excluded from the study because of incomplete consent or for failing to return the questionnaire. Fourteen students were excluded because of an inadequate amount of urine submitted for analysis.
Written informed consent was obtained from the parents of all participants and ascent was obtained from all students older than 7 years after they were informed of the study protocol and procedures including the urine specimen collection process. The study was approved by the Institutional Review Boards of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (IRB 050/56).
All data were collected at the date of urine collection. The questionnaire was divided into 2 parts, which included demographic data and possible exposure risks. Demographic data included age, sex, current residence, eating habits, caregiver education, family income, awareness of pubertal status, and total daily hours of television watching. BPA exposure risks included junk food, canned food, and bottled milk consumption, use of plastic containers for food and
water, use of microwave to heat plastic food containers and plastic toys. The frequency of exposure was classified into 6 categories, including “never”, “at least once per month”, “once per month”, “once per week”, “more than 2 days per week” and “at least once a day”.
Body weight (kg) and height (cm) were measured by trained teachers. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m2). Age- and sex-standardized BMI
Morning urine samples (40-50 mL) were obtained from students in BPA free polypropylene tubes. The collected urine samples were immediately transported to a laboratory and stored at –80°C until analysis [43].
For each urine sample, total urinary BPA concentrations (free plus conjugated species) were analyzed by solid-phase extraction (SPE) coupled with high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS). The lower limit of quantification (LLOQ) of BPA was 0.05 ng/mL.
Urine samples (500 μL) were fortified with 50 ng/mL of d16 -BPA as an internal standard and supplemented with 100 μL of ammonium acetate buffer (0.1 M, pH 6.5) and 1,200 units (12 μL) of glucuronidase/sulfatase. The mixture was incubated at 37°C for 3 h and then centrifuged at 13,000 rpm for 10 min before clean up by solid-phase extraction (SPE) using an Oasis HLB cartridge (60 mg/3 mL; Waters, Milford, MA). Cartridges were preconditioned with 5 mL of methanol and 5 mL of deionized (DI) water. After loading samples, the cartridge was washed with 2 mL of 0.1 N HCl and 5 mL of 10% methanol in water and eluted with 3 mL of methanol. The elute was concentrated to 150 μL using centrifugal vacuum concentrator (MiVac, Genevac Inc, NY, USA) and subjected to HPLC–MS/MS analysis.
Quantitation of BPA was based on calibration curves of 8 concentration points: 0.05, 0.1, 0.5, 1, 5, 10, 20, and 50 ng/mL obtained after fortification with d16 -BPA as the internal standard followed by extraction using the same method as described for the preparation of urine samples.
Quantification of BPA was performed using an Agilent 6490 HPLC-MS/MS (Agilent Technologies Inc, Santa Clara, CA). The chromatographic column used was Zorbax RRHD Eclipse Plus C18 (2.1 mm × 50 mm, 1.8 μm; Agilent Technologies Inc). The mobile phase was 0.05% acetic acid in DI water and acetonitrile (60:40). The system was run with an isocratic elution at 0.4 mL/min. The column temperature was 30°C and injection volume was 4 μ
Urine creatinine (Cr) was analyzed using the Jaffe method with a Beckman Coulter reactive in AU5400 (IZASA) (Beckman-Coulter Inc). Each urinary BPA concentration in ng/mL was adjusted to g/g Cr.
Daily intake of BPA was estimated using the following equation; Daily intake ( μg/day) = Urinary BPA concentration ( μg/L) × urine excretion rate (L/day). Daily urinary excretion rates were reported to be 500, 700, and 1,200 mL for ages below 5, 6-11, and 12-18 years, respectively [44].
A chi-squared test was used to evaluate dichotomized variables, while an unpaired
We included 376 participants in the study (193 boys and 183 girls). Their mean age was 10.36 ± 3.39 years (range 3.58-17.17 years). BPA was detected at a level of more than 0.05 ng/mL in 283 (75.3%) of the 376 urine specimens. The overall median concentration of urinary BPA was 0.68 ng/mL (IQR 0.06-1.48; maximum concentration of 8.75) and the median concentration of creatinine-adjusted BPA concentration was 0.53 (IQR 0.04-1.12). In boys, the median concentrations of BPA and adjusted BPA were 0.78 (0.23-1.72) ng/mL and 0.54 (0.151.18) μg/g Cr, respectively. In girls, the median concentrations of BPA and adjusted BPA were 0.59 (0-1.31) ng/mL and 0.5 (0-1.02) μg/g Cr, respectively (
Urinary bisphenol A (ng/mL), creatinine adjusted bisphenol A (μg/g Cr), and estimated daily intake (μg/day) by ages, genders, family income, and body mass index
| Characteristics | Adjusted urinary bisphenol A (μg/g Cr) | Estimated daily intake (μg/day) | ||
|---|---|---|---|---|
| Mean ± S D | Median (IQR) | Mean ± S D | Median (IQR) | |
| 3.01-6.00 | 1.21 ± 1.53 | 0.70 (0-1.90) | 0.55 ± 0.79 | 0.28 (0-0.66) |
| 6.01-10.00 | 0.97 ± 1.43 | 0.71 (0-1.10) | 0.67 ± 0.86 | 0.43 (0-0.87) |
| 10.01-18.00 | 0.78 ± 1.07 | 0.45 0.13-0.98) | 1.42 ± 1.79 | 0.80 (0.18--2.04) |
| 0.90 ± 1.28 | 0.53 (0.04-1.12) | 1.04 ± 1.46 | 0.55 (0.06-1.48) | |
| Male | 0.90 ± 1.09 | 0.54 0.15-1.18) | 1.15 ± 1.49 | 0.60 (0.20-1.54) |
| Female | 0.91 ± 1.45 | 0.50 (0-1.02) | 0.92 ± 1.41 | 0.44 (0-1.09) |
| <453 | 0.88 ± 1.15 | 0.53 (0.04-1.14) | 0.99 ± 1.36 | 0.55 (0.040-1.25) |
| 453-906 | 0.74 ± 0.97 | 0.41 (0-0.97) | 1.09 ± 1.86 | 0.35 (0-1.43) |
| >906 | 1.59 ± 2.44 | 0.71 (0.35-0.31) | 1.46 ±1.35 | 0.76 (0.41--.34) |
| Normal weight | 0.91 ± 1.35 | 0.51 (0-1.09) | 0.98 ± 1.39 | 0.50 (0-1.32) |
| Overweight | 0.65 ± 0.96 | 0.36 (0-0.76) | 1.14 ± 2.18 | 0.45 (0-1.03) |
| Obese | 0.90 ± 1.03 | 0.71 (0.21.16) | 1.24 ± 1.43 | 0.65 (0.30-0.04) |
The mean estimated daily exposure of BPA was 1.04 ± 1.46 μg/day as shown in
The prevalence of overweight and obese children in this study were 9% and 11.3%, respectively. Detection rates were higher in children 3-9 years of age than in the adolescents, and higher in obese children than those that were not obese (
Bisphenol A detection by age, sex, family income, and body mass index
| n (%) | Urinary bisphenol A (n, %) | |||
|---|---|---|---|---|
| <0.05 ng/mL | ≥0.05 ng/mL | |||
| 3.01-6.00 | 40 (10.6) | 15 (16.2) | 25 (32.5) | 0.048 |
| 6.01-10.00 | 146 (38.8) | 39 (41.9) | 29 (37.7) | 0.479 |
| 10.01-18.00 | 190 (50.6) | 39 (41.9) | 23 (29.8) | 0.056 |
| Male | 193 (51.3) | 41 (44.1) | 152 (53.7) | 0.121 |
| Female | 183 (48.7) | 52 (55.9) | 131 (46.3) | 0.121 |
| <453 | 273 (74.0) | 68 (73.9) | 205 (74.0) | 0.899 |
| 453–906 | 67 (18.2) | 23 (25.0) | 44 (15.9) | 0.045 |
| >906 | 29 (7.8) | 1 (1.1) | 28 (10.1) | 0.006 |
| Normal weight | 274 (79.7) | 77 (86.5) | 197 (77.3) | 0.015 |
| Overweight | 31 (9.0) | 8 (9.0) | 23 (9.0) | 0.831 |
| Obese | 39 (11.3) | 4 (4.5) | 35 (13.7) | 0.030 |
Association between body mass index, family income, and detection of bisphenol A in urine (%)
| OR | 95% Confidence interval for OR | ||
|---|---|---|---|
| <453 | 1.00 | 1.00 | |
| 453–906 | 0.64 | 0.36–1.13 | 0.12 |
| >906 | 9.29 | 1.24–69.56 | 0.03 |
| Normal weight | 1.00 | 1.00 | |
| Overweight | 1.12 | 0.48–2.62 | 0.79 |
| Obese | 3.42 | 1.18–9.95 | 0.02 |
Urinary bisphenol A level association with body mass index
| Body mass index | |||||
|---|---|---|---|---|---|
| Normal (n = 724) | Overweight (n = 31) | Obese (n = 39) | Normal vs overweight | Normal vs obese | |
| Urinary bisphenol A (ng/mL) | 0.67 | 0.62 | 0.87 | 0.63 | 0.14 |
| Adjusted bisphenol A (μg/g Cr) | 0.51 | 0.36 | 0.71 | 0.30 | 0.19 |
To our knowledge, this is the first report of urinary BPA concentrations in Thai children and adolescents. The detection rate of BPA was 75.3% with a median concentration of 0.68 ng/mL. Sriphrapradang et al. reported the detection rate of BPA in Thai adults as 52.8% with a median serum BPA concentration of 0.33 ng/mL as determined by ELISA [17]. Although interesting, those findings cannot be compared directly with those in the present study because of the different samples and techniques used. However, our data suggest that BPA exposure may be higher in Thai children than in adults. A limitation of analyzing BPA in serum is the rapid degradation and elimination of BPA [40]. Therefore, urinary samples may provide more information regarding chronic BPA exposure [41]. The present study showed that Thai children and adolescents had lower levels of urinary BPA (0.68 vs 0.70-1.70 ng/mL) and detection (75.3% vs 79%-94.3 %) compared with data from Northern America, some European, Egypt, and other Asian countries [7-16]. The lowest observable adverse effect level was set in the 1980s at 50 mg/kg/day [45]. The European Food Safety Authority and U.S. Environmental Protection Agency (EPA) use the same “reference dose” or safe dose of 50 mg/kg/day [44] and this is calculated using the following formula “daily intake in μg/day is equal to urinary BPA concentration in μg/L × urine excretion rate in L/day”. Daily urine excretion rates were reported to be 500, 700, and 1,200 mL for ages younger than 5, 6-11, and 12-18 years, respectively [44]. The median daily BPA intake was 0.55 (IQR 0.06-1.48) μg/day, which was lower than the reference safe dose by nearly a million fold. However, various molecular studies have shown a variety of pathways in which BPA can stimulate cellular responses at very low doses and where the effects can be U-shaped or inverted U-shaped dose responses, so-called “nonmonotonic” association [15-47].
Increasing numbers of studies in humans showed that a higher concentration of urinary BPA is associated with younger age [7, 14], female sex, non-Hispanic black, low family income [7], low education, and smoking [13, 27]. BPA can leach from polycarbonate plastic food containers and BPA-coated metallic cans. Urinary BPA levels are reported to be associated with food in plastic containers [16], bottled milk feeding [3], and eating canned food [13]. However, urinary BPA levels in the present study were not related to sex, family income, or perceived dietary behavioral risk of BPA exposure. BPA detection, but not its levels, were more significantly associated with children aged 3-6 years than children aged 6-10 years, and adolescents. Our results are consistent with most published studies, which postulate that children consume more food per body weight and are more vulnerable to exposure to products with BPA contamination such as plastic packages and toys [7, 12, 14].
We showed an association between obesity and urinary BPA detection. The BPA detection rate in students with normal weight was significantly higher in the group with BPA <0.05 ng/mL than in the group with BPA >0.05 ng/mL (86.5% vs 77.3%,
Literature reporting the association of bisphenol A and obesity in children and adolescents
| Authors | Country | Year | Ν | Age | Method | Limit of detection (ng/mL) | Bisphenol A (ng/mL) | Obesity (%) | Over-weight (%) | Bisphenol A (ng/mL) and obesity | |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 2011 Carwile XL, et al [24] | USA | 2003-2006 | 2,747 | 18-74 | LCMS | 0.3 | 2.05 (median) | 33.9 | lstQ(BPA<l.l) | Significant, 95%CI | |
| 2nd Q (1.2-2.3) | |||||||||||
| 3rd Q (2.4–1.6) | |||||||||||
| 4th Q (>4.7) | |||||||||||
| OR 1.60-1.85 | |||||||||||
| 2012 Trasande L, et al [25] | USA | 2003-2008 | 2838 | 6-19 | LCMS | 0.3 | 2.8 (median) | 17.8 | 34.1 | lstQ(BPA≤1.5) | < 0.001 |
| 2nd Q (1.5-2.7) | |||||||||||
| 3rd Q (2.8-5.5) | |||||||||||
| 4thQ(>5.6) | |||||||||||
| OR 2.08-2.57 | |||||||||||
| 2012 Bhandari R, et al [26] | USA | 2003-2008 | 2200 | 6-18 | LCMS | 0.3 | 4.8 | 17.7 | 16.7 | lstQ(BPA≤1.5) | 0.002 |
| 2nd Q (1.5-2.7) | |||||||||||
| 3rd Q (2.8-5.4) | |||||||||||
| 4thQ(>5.4) | |||||||||||
| OR 1.78-2.55 | |||||||||||
| 2012 Wang H, et al [27] | China | 2011 | 259 | 8-15 | LCMS | 0.07 | 0.45 GM | 31.7 | 20.4 | Normal BPA 0.33 Obesity BPA 0.57 | 0.018 |
| 2013 Li D, et al [29] | China | 2011 | 1,326 | 4-12 | HPL/FD | 0.31 | NA | 18.3 | girl: associated with overweight | Significant, 95%CI | |
GM = geometric mean, HPL/FD, HPLC with fluorescence detection, NA = not available, Q = quartile
There have been several hypotheses postulated to date for the mechanism of BPA exposure leading to obesity [48]; but the details remain unclear. In vivo studies in mice show that exposure to low doses of BPA during the perinatal development, resulted in increased body weight [49, 50], and BPA was identified as associated with the upregulation of a number of genes in abdominal adipocytes in rats, including those for peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, and lipoprotein lipase [51]. In vitro studies showed that BPA acts as a selective estrogen receptor moderator, which can then regulate adipocyte hypertrophy and hyperplasia [52]. BPA was found to stimulate glucocorticoid receptors to promote adipogenesis in preadipocytes (3T3-L1 cell line) [53]. To our knowledge, there is no published study showing increased BPA absorption in obese individuals.
The present cross-sectional study is limited by its small sample size and because sampling was not conducted using a randomized method. A number of questionnaires were not completed, thereby, perhaps affecting the data. This might have been because participants could not understand some of the questions. Therefore, future questionnaires will have to take this into consideration. Urine collection was difficult in small children, particularly from girls. Spot morning urine is not as reliable as 24 hour urine samples or multiple spot urine testing, but it might be acceptable in a larger study. A strength of our study is use of a highly sensitive and standardized LC-MSMS method to analyze urine BPA.
BPA exposure in Thai children and adolescents appears to be lower than that in Northern America, some European, and other Asian countries. Obesity and younger age are significantly associated with BPA detection.