Article pubs.acs.org/est
Temporal Changes of Urinary Oxidative Metabolites of Di(2ethylhexyl)phthalate After the 2011 Phthalate Incident in Taiwanese Children: Findings of a Six Month Follow-Up Chia-Fang Wu,† Bai-Hsiun Chen,‡,§ Jentaie Shiea,∥ Eric K. Chen,⊥ Ching-Kuan Liu,# Mei-Chyn Chao,§ Chi-Kung Ho,† Jiunn-Ren Wu,§ and Ming-Tsang Wu*,†,∇,○ †
Department of Public Health, College of Health Sciences, Kaohsiung Medical University, Room 721, CS Building, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan ‡ Department of Laboratory Medicine, Kaohsiung Medical University Hospital, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan § Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan ∥ Department of Chemistry, National Sun Yat-Sen University, No.70, Lienhai Road, Kaohsiung 804, Taiwan ⊥ Center for General Education, Kaohsiung Medical University, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan # Department of Neurology, Kaohsiung Medical University Hospital, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan ∇ Department of Family Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, No.100, Shih-Chuan First Road, Kaohsiung 807, Taiwan ○ Center of Environmental and Occupational Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, No.482, Shanming Road, Kaohsiung 812, Taiwan S Supporting Information *
ABSTRACT: A major incident involving phthalates-contaminated foodstuffs occurred in Taiwan in May 2011, leading to the quick removal of tainted food items from store shelves. We investigated changes in urinary oxidative di(2ethylhexyl)phthalate (DEHP) metabolites, our proxy for exposure to DEHPtainted foodstuffs in children ≤10 years, during the six months following withdrawal of the tainted food. Our hospital screened 60 possibly exposed children between May and June 2011. The children’s food intake information was collected, and they were administered one-spot urine samples at baseline and at the two and six month follow-ups. All three samples were measured for four oxidative DEHP metabolites, mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP), and mono-(2-carboxymethylhexyl) phthalate (2cx-MMHP) by triple quadrupole liquid chromatography tandem mass spectrometry. Fifty-two children had been exposed. After excluding those without a full set of urine samples or adequate food intake information, 23 exposed children were studied. We found significantly positive correlations between DEHP daily intake and urinary 5OH-MEHP, 5oxo-MEHP, and 5cx-MEPP (p < 0.05). At the six month follow-up, all four metabolite concentrations had significantly decreased compared to the baseline. In conclusion, urinary DEHP metabolites decreased progressively in children after tainted food withdrawal, indicating that the main sources of phthalate contamination for children had been successfully controlled.
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INTRODUCTION
endocrine function and adversely affect sex and thyroid hormones, the reproductive function, and neurodevelopment.3−5 Because DEHP is more toxic than DINP and most of the foodstuffs had been contaminated by DEHP in this
A major incident of phthalate-contaminated foodstuffs occurred in Taiwan between April and July, 2011.1,2 Phthalates, mainly di-(2-ethylhexyl) phthalate (DEHP) and/or Di-isononyl phthalate (DINP), had been deliberately added to foodstuffs as a substitute of emulsifier in five major food categories, one being nutrient supplements regularly taken by children.1,2 Phthalates such as DEHP and DINP, which are commonly added to plastics to increase flexibility, are thought to disrupt © 2013 American Chemical Society
Received: Revised: Accepted: Published: 13754
April 1, 2013 November 5, 2013 November 5, 2013 November 5, 2013 dx.doi.org/10.1021/es403141u | Environ. Sci. Technol. 2013, 47, 13754−13762
Environmental Science & Technology
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incident, the Taiwan Food and Drugs Administration (TFDA) provided more information about the concentration of DEHP than DINP in affected foods on its official Web site.2,6,7 The authorities had all foods identified as being tainted removed from the market by May 31, 2011. It was not clear whether this policy adequately removed the threat. In order to answer that question, this study first investigated the association between a questionnaire reporting DEHPtainted food intake data and urinary concentration of oxidative DEHP metabolites data collected from prepuberty children ≤10 years. Findings were confirmed by comparing DEHP daily intake estimated from questionnaire data and from urinary oxidative DEHP metabolites concentration. We then measured urinary oxidative DEHP metabolites at two and six months to follow changes in DEHP exposure in these children to find out if the government’s removal of the specific food products adequately resolved the public health threat.
Figure 1. Study flowchart.
MATERIAL AND METHODS Study Subjects. At a Phthalates Clinic for Children (PCC) newly established at Kaohsiung Medical University Hospital, children potentially exposed to phthalates-tainted foodstuffs were screened between May 31 and June 17, 2011, following a design described in detail elsewhere.8 Briefly, all children who visited this special clinic and whose parents were willing to participate in this study were included. Sixty children who were ≤10 years and had not received any hormone treatment served as the potential study subjects. Food intake information was obtained from questionnaire by interviewing the main caregivers, particularly mothers, of the children, and the children provided urine samples at baseline and at two and six months for analyzing oxidative DEHP metabolites represent as DEHP exposure. This study was approved by the Institutional Review Board of KMUH. Informed written consent was provided by the parents of the studied children. Exposure Information on Affected Foodstuffs. The parents, mostly mothers, of the potential study children were interviewed by two trained interviewers to collect the exposure information about intake of phthalates-tainted food items, defined as previously being found to have DEHP or DINP ≥ 1 ppm.2,8 The exposed children were those exposed to any contaminated food item listed in the official Web site of TFDA and in the tainted-food data set from Kaohsiung Bureau of Health.2 Non-exposed children were those not exposed to any listed tainted-food items (Figure 1). DEHP concentrations for these food items were collected from official reports from the Taiwan Food and Drug Administration and Bureau of Health of Kaohsiung City.2,8 These concentrations along with reported intake of these foods were used to construct daily intake dose (μg/kg body weight (bw)/day) among the potential study children. Daily intake was converted from the sum of exposure amount (μg per time) and frequency (times per day) divided by body weight (kg) of each child. For example, assuming one child weighing 12 kg ate Power-Lac Nutrition Powder (527.0 μg/g of DEHP) for 2 g per pack per day,2 we estimated his daily intake of DEHP would be 87.83 μg/kg bw/day ((527 μg/ g × 2 g/day)/12 kg)). Collection of Urine Specimens during Six Month Follow-Up. One-spot urine samples were collected from all potential study children at baseline during their first visit to the PCC. One-spot urine samples were also collected at the two and six month follow-ups. All urine specimens were collected in a 15 mL polypropene (PP) tube (cat. no. 352097, BD Falcon,
NJ, U.S.A.), divided into 2 mL PP tube (Part# 2340-00, Scientific Specialties, Inc., Lodi, CA) and stored at −20 °C until analysis. Analysis of Oxidative DEHP Metabolites in Urine. Sample Preparation. Oxidative DEHP metabolites in urine were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) according to the analytical method used by Silva et al.9 Briefly, 1 mL urine sample was thawed, transferred to a glass tube, added to a 13C4-5OH-MEHP and 13 C4-5oxo-MEHP-labeled solution as an internal standard (Cambridge Isotope Laboratories, Andover, MA, U.S.A.), and then buffered with 250 μL ammonium acetate (1M, pH 6.5) and β-glucuronidase enzyme (from E. coli, K12, Roche Biomedical, Mannheim, Germany). The samples were incubated in a 37 °C water bath for 90 min. After hydrolysis, each sample was acidified by adding 2 mL phosphate buffer (0.14 M NaH2PO4 in 0.85% H3PO4), vortex-mixed, and centrifuged at 3500 rpm for 10 min. The supernatant was loaded into a solid-phase extraction cartridge (NEXUS, Varian, Inc., Palo Alto, CA, U.S.A.). Formic acid (2 mL) and water (2 mL) were added to the cartridge to remove hydrophilic compounds, and then acetonitrile (1 mL) and ethyl acetate (1 mL) were added to elute oxidative DEHP metabolites. The combined elutes were concentrated under a stream of dry nitrogen at 55 °C. Finally, the residues were reconstituted with water and subjected to LC-MS/MS for analysis. To avoid the potential contamination with phthalates during sample collection and preparation, we followed a method used by Huang et al. with slight modification.10 Before glassware samples were used, they were first washed with neutral detergent, followed by water, acetone, acetonitrile, and methanol, each sonicated for 15 min, and finally rinsed with methanol. The glassware was then dried using nitrogen. HPLC grade water was used to prepare aqueous buffers for the mobile phase. All other reagents were also analytical grade or better. LC-MS/MS. LC-MS/MS analysis was performed using an Agilent 1200 HPLC (Agilent Technologies, Palo Alto, CA, U.S.A.), which is coupled to a API 4000Q triple-quadrupole mass spectrometer (API 4000Q, Applied Biosystems/MDS SCEX, Concord, Canada) equipped with an electrospray ionization (ESI) source in a negative ion mode. A 10 μL sample was injected into a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent) at a flow rate of 1000 μL/min in the gradient mode from 80% mobile phase A (0.1% acetic acid in
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addition, the 12 children have not been exposed to any phthalates-tainted food. Before use, the glass cups were washed and rinsed as mentioned above. None of the four oxidative DEHP metabolites were detected in the pre-sampling glass cups. After one-spot urine samples were collected from the 12 children in these clean glass cups, the cups were immediately covered with aluminum foil and transferred to the laboratory. Before analysis, each urine sample was halved with one-half kept in the glass cup and the other half poured into a PP tube to simulate the same collection process used to collect urine samples from our potential study children. Urine samples in glass cups and PP tubes were simultaneously analyzed for the four oxidative DEHP metabolites. Estimated DEHP Daily Intake from Urinary Oxidative DEHP Metabolites. DEHP daily intake in each study child was also estimated based on the creatinine excretion-based model from the four urinary oxidative DEHP metabolites.11 The creatinine excretion-based model was followed on the following equation
water) to 90% mobile phase B (0.1% acetic acid in acetonitrile). The TurboIon-Spray source was run at a temperature of 550 °C using the following settings: curtain gas, 25 (arbitrary units); source gas 1, 50; source gas 2, 55; CAD gas pressure, medium; and ion spray voltage, 5500. The precursor ion (m/z), production ion (m/z), collision energy (eV), and retention times (min) of the four oxidative DEHP metabolites and the two internal standards were as follows: 293/121/−26/7.2 for 5OH-MEHP, 291/121/−28/7.6 for 5oxo-MEHP, 307/159− 18/6.7 for 5cx-MEPP, 307/159−18/7.2 for 2cx-MMHP, 297/ 124/−28/7.2 for 13C4-5OH-MEHP, and 295/124/−24/7.6 for 13 C4-5oxo-MEHP (Figure 1, Supporting Information). Method Validation. The calibration was performed using standard solutions of DEHP oxidative metabolites in pooled urine samples. The corresponding ring-labeled analogs were used as internal standards. Because internal standards of 5cxMEPP and 2cx-MMHP were not available, calibration of these analytes were performed using the internal standard of 5oxoMEHP. The calibration range of each metabolite was divided into two: 1−50 ng/mL for the low one and 50−1000 ng/mL for the high one. The correlation coefficients (R2) of these calibrations were 0.9998 for 5OH-MEHP, 0.9998−0.9999 for 5oxo-MEHP, 0.9970−0.9975 for 5cx-MEPP, and 0.9992− 0.9995 for 2cx-MMHP, all of which were higher than 0.9950. Internal quality control was performed by analyzing both a low (10 ng/mL) and a high (100 ng/mL) level spiked into the urine in each batch. The accuracies for all calibration concentration curves and internal quality controls ranged from 87.5 to 110.1% and had precisions expressed as a coefficient of variance (CV) ranging from 1.3 to 6.8% (n = 5). The intra- and inter-day relative standard deviations (RSD) ranges were 1.50−5.40% and 0.16−8.12% respectively. The averaged internal standard recoveries in urine mixtures ranged from 88.5 to 90.0% ( Table 1, Supporting Information). In addition to performing each urine analysis, we analyzed 1−2 reagent blanks, one each for low and high concentration quality control samples, and 1−2 previously analyzed samples whose values were known to confirm our findings. A calibration check was also run every 20 samples to ensure instrumental stability throughout the entire analysis. Quantification of the calibration concentrations was within 15% of the theoretical value with a CV less than 15%, meeting performance criteria. The limits of quantification (LOQ), defined as a signal-to-ratio of six within accuracy 100 ± 15%, were 1 ng/mL for the four oxidative DEHP metabolites. The method of detection limit (MDL), determined using a urine sample spiked with standards, was 0.2 ng/mL for the four oxidative DEHP metabolites (Table 1, Supporting Information). Any measurement below MDL was treated as half-MDL. For urinary creatinine, a spectrophotometer (U-2000, Hitachi, Tokyo, Japan) was used to measure the creatinine− picrate reaction at a wavelength of 520 nm. Because we also corrected for urinary creatinine, measurements of urinary oxidative DEHP metabolites were expressed as μg/g creatinine. Urine Collection from Additional 12 Volunteer Children. In order to confirm that the oxidative metabolites of DEHP were not contaminated by the urine container or the external environment during the urine specimen collection, we first prepared clean glass cups to collect one-spot urine samples from another 12 volunteer and healthy children as controls. The 12 children were the children of our hospital staff members who were willing to participate in this study, and their ages were similar to those of our potential study children. In
DEHP(μg/kg/day) = [UEsum(μmol/g Cr) × CEsmoothed(g Cr/day)/FUE × BW(kg)] × MWDEHP
where UEsum, CEsmoothed, FUE, and MWDEHP represented the molar urinary excretion sum of the four measured urinary oxidative DEHP metabolites, smoothed creatinine excretion rates, molar fraction, and molecular weight of DEHP, respectively. According to the study of Remer et al.,12 CEsmoothed was calculated based on body height- and genderbased reference values for urinary creatinine excretion in healthy white children aged 3−18 years. FUE for the four oxidative DEHP metabolites was set as 0.61 based on a study by Koch et al.13 Statistical Analysis. For the 12 healthy controls, a Wilcoxon signed rank test was used to examine the absolute differences of each urinary oxidative metabolite level between the two groups (glass cup and PP groups). For the potential study children, median (interquartile range, IQR) or number (frequency) was used to describe demographic characteristics and urinary metabolites when appropriate. Mann−Whitney U test was used for continuous variables and Fischer’s exact test for categorical variables used to compare children with and without urine samples for the analyses of DEHP metabolites. Among the exposed children with known DEHP concentration of affected foods, Spearman correlation was used to examine the association between DEHP daily intake and each urinary metabolite at baseline and the association of estimated daily intake calculated from questionnaires and from urinary DEHP metabolites concentrations. At follow-up, Wilcoxon signed rank test was used to compare the absolute difference of each urinary metabolite. Data were analyzed using the SAS version 9.2 (SAS Institute Inc., Cary, NC); all p-values were two-sided with significance set at 0.05, Wilcoxon signed rank test) (Figure 2(b), Supporting Information). Baseline Oxidative DEHP Metabolites Levels in the Study Children. Thirty-seven of the 60 potential study children provided enough urine for analyses and the measurement of the four oxidative metabolites at baseline (Figure 1). There was no significant difference in age, gender, and body weight between children with (n = 37) and without urine samples (n = 23) for analyses (Table 2, Supporting Information). According to the information from the parents of the study children, 32 of the 37 had been exposed to affected foods at least once. Among the 32 exposed children, 12 stopped eating any affected food before the date of the public release of this food scandal by news (May 25, 2011), 14 stopped eating any affected food after May 25, 2011, and the remaining 6, whose parents could not remember the exact date of stopping, also ceased eating any affected food. The median delayed days (range) when urine specimens were collected at baseline were 1029 (194−7204) days for those 12 children and 6 (1−12) days for those 14 children (data not shown). Among the 32 exposed children, the actual concentrations of DEHP in their affected foods were known in 23, making it possible to calculate their daily intake. Of these 23 children, the 13757
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to the baseline (Wilcoxon signed rank test, all p-values
