|Year : 2019 | Volume
| Issue : 1 | Page : 6-11
Bisphenol A (BPA) in liquid portions of canned foods obtained from domestic and Asian markets in the United States
Aby Joiakim1, David Kaplan1, David A Putt1, Julia Matzenbacher Santos2, Klaus Friedrich3, So Hee Kim4, Hyesook Kim5
1 Detroit R&D, Inc., Detroit, MI, USA
2 Detroit R&D, Inc., Detroit, MI; Education, Health and Human Performance Department, Fairmont State University, Fairmont, WV, USA
3 Chemistry and Biochemistry Department, University of Detroit Mercy, Detroit, MI, USA
4 College of Pharmacy and Research Institute of Pharmaceutical Science and Technology, Ajou University, Suwon, Korea
5 Detroit R&D, Inc; Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA
|Date of Submission||25-Jan-2019|
|Date of Acceptance||18-Feb-2019|
|Date of Web Publication||9-Apr-2019|
Dr. Hyesook Kim
Detroit R&D, Inc., 2727 Second Ave. Suite 4113, Detroit MI 48201
Source of Support: None, Conflict of Interest: None
Bisphenol A (BPA) is a phenolic environmental estrogen that disrupts endocrine activity thereby increasing the risk of hormone-related health problems. The human population is highly exposed to BPA and food is believed to be a primary source of BPA exposure. The aim of this study was to test the sensitivity and specificity of a BPA enzyme-linked immunosorbent assay (ELISA) and to measure levels of BPA in supernatants obtained from various canned foods from different countries. The concentration of BPA was measured in supernatant from different types of canned soup and vegetable mixes produced by US companies and two companies each from three different Asian countries (Korea, Japan and China), which are available at markets in the USA. ELISA results were confirmed by LC/MS/MS and shown to be in agreement. Cross-reactivity tests demonstrated that BPA ELISA kit does not cross-react with other tested phenolic compounds. There was no significant difference of BPA levels in different types of soups from different US companies. However, levels of BPA in supernatants of canned vegetable mixes of a company in the USA were 200-fold lower than the levels in canned vegetable soups of the US companies. BPA levels varied greatly among canned foods among companies in various countries. Thus, this study validated the use of a simple ELISA assay to measure levels of BPA in supernatants of canned food, which would facilitate the routine monitoring of dietary exposure to BPA. Decreasing the consumption of BPA will lead to a reduction in the risk of adverse health effects.
Keywords: Bisphenol A, canned food, endocrine disruptor, environmental estrogen
|How to cite this article:|
Joiakim A, Kaplan D, Putt DA, Santos JM, Friedrich K, Kim SH, Kim H. Bisphenol A (BPA) in liquid portions of canned foods obtained from domestic and Asian markets in the United States. Environ Dis 2019;4:6-11
|How to cite this URL:|
Joiakim A, Kaplan D, Putt DA, Santos JM, Friedrich K, Kim SH, Kim H. Bisphenol A (BPA) in liquid portions of canned foods obtained from domestic and Asian markets in the United States. Environ Dis [serial online] 2019 [cited 2022 May 22];4:6-11. Available from: http://www.environmentmed.org/text.asp?2019/4/1/6/255739
| Introduction|| |
Bisphenol A (BPA) is a phenolic environmental estrogen that mimics the action of 17β-estradiol (E2) in mammals disrupting endocrine activity. BPA increases the risk of hormone-related health problems such as infertility, breast, ovarian and prostate cancers, Parkinson's disease and insulin resistance.,,,, Even with all these risk factors, over 8 billion pounds of BPA are continuing to be used in the manufacturing of phenolic polymers and epoxy resins for metal linings of canned food and beverage containers over a year.
The human population is highly exposed to BPA and studies have reported high levels of BPA in human urine as well as in sewage water.,,,, Food is believed to be the primary source of BPA exposure in humans since BPA can migrate from the epoxy resin lining of a can to the contents of the can. Air, dust, thermal paper, and water also contribute to human exposure to a lesser extent.,,, BPA levels of canned solid food have been measured in an important study performed by the Food and Drug Administration (FDA) laboratory using liquid chromatography-tandem mass spectrometry (LC/MS/MS) after extraction and levels ranged from 13 to 110 ng BPA/g. In agreement, a great number of studies have demonstrated a high level of BPA in different types of canned food and beverages even after legislation limiting the use of BPA in food packaging.,,, Altogether, these reports show the importance of the continued monitoring of dietary exposure to BPA.
Therefore, an assay that can easily measure BPA without the need of extraction would be of great benefit to the large scale control of BPA in canned food. Thus, a form-specific competitive enzyme-linked immunosorbent assay (ELISA) for BPA will facilitate environmental screening for BPA in dietary and non-dietary materials. The aim of this study was to test a commercial ELISA for specificity and sensitivity and to measure levels of BPA in supernatants obtained from various canned foods produced in the USA and Asian countries, which are available in US markets.
| Materials and Methods|| |
BPA (97% purity) was obtained from Alfa Aesar (Wood Hill, MA). The BPA antibody was produced in goat. Bisphenol F and S (BPF and BPS) and resveratrol were obtained from Alfa Aesar and Sigma-Aldrich (St. Louis, MO), respectively, to test for cross-reactivity of the BPA antibody with similar phenolic compounds. The commercially available BPA-ELISA (Detroit R&D, Inc, Detroit, MI) was tested using concentrations of BPA, BPF, BPS and resveratrol ranging from 10 pg/ml to 1 μg/ml. The cross-reactivity to these other molecules was determined by comparing their concentration at 50% B/B0 to that of BPA. The specificity of the antibody was also measured by immunoblot assay. First, the diacyclohexyl carbodiimide (DCC) method (ThermoFisher Scientific, Waltham, MA) was used to conjugate bovine serum albumin (BSA) to BPA. Immunoblot analysis was performed using anti-serum against BSA or BSA conjugated with BPA (BPA-BSA) to test for cross-reactivity of the goat antibody to BPA. Horseradish peroxidase (HRP) conjugated anti-goat antibody (Detroit R&D, Inc) and enhanced chemiluminescent (ECL) reagents (Detroit R&D, Inc) were used to visualize the binding of BPA with antibody.
To test the sensitivity of the ELISA method, soup samples were spiked with 10 ng of BPA. The liquid portion of the canned soup was centrifuged at 1,500 ×g for 10 min followed by 18,000 ×g for 10 min to eliminate suspended pieces. Then 5 μl dimethylformamide (DMF), with or without 10 ng BPA, was added to 1 ml of supernatant. The supernatant was diluted 10-fold with high performance liquid chromatography (HPLC)-grade water and the competitive ELISA assay (Detroit R&D, Inc.) was carried out using BPA conjugated to HRP as the competing molecule.
Bisphenol A assessment of supernatants obtained from canned foods
Different types of canned soups, low sodium minestrone, minestrone and vegetable (n = 3, each), produced in US were obtained from US domestic food markets (Country #1 Company A). In addition, canned foods from two companies in each of three different Asian countries (Country #2, #3 and #4) were also obtained from US and Asian markets (3 cans each). Canned vegetable mix (n = 3 cans) and canned vegetable soup (n = 2 cans) from different US companies were also tested. Products were purchased between 2012 and 2014. Supernatants of canned foods were collected and centrifuged at 1,500 × g followed by 18,000 × g for 10 min each. The pH of the supernatant was measured and the supernatant was diluted 10-fold with sample dilution buffer (pH 7.5). Competitive ELISA was performed in triplicate for each food sample.
Determination of bisphenol A in soups by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Soup samples obtained from two different companies in US (Companies A and B) and standards were prepared in 1:1 methanol/water and analyzed using a Shimadzu Nextera X2 UPLC-MS/MS equipped with a Waters 2.1 × 50 mm, 1.7 μm ultra-performance (UP) LC (Waters Corporation, Milford, MA) BEH C18 column at the Lumigen Instrument Center, Wayne State University (Detroit, MI). The retention time of BPA was approximately 2.01 min when using a linear gradient of 10 to 100% acetonitrile in water and an oven temperature of 45°C. The injection volume was 20 μl. The SRM transitions used were m/z 227–212 for the qualifier and m/z 227–133 for the quantifier. Shimadzu Lab Solution v. 5.65 software was used for data analysis.
Statistical analyses were performed using commercially available software (SigmaSalt). To test for normal distribution of data sets, we used the Shapiro-Wilk test. The Mann-Whitney U test was used for statistical analysis for two groups' comparison. The Kruskal-Wallis test and the Dunn test were used for analysis of data among more than two groups. Data are expressed as mean ± standard deviation (SD) and P < 0.05 was considered statistically significant.
| Results|| |
Antibody and enzyme-linked immunosorbent assay quality control
The cross-reactivity tests with BPF (no methyl group) and BPS (methyl group substituted with a sulfone) demonstrated minimal cross-reactivity of the anti-BPA antibody with BPF (2%), BPS (<0.01%) or resveratrol (<0.01%), which indicates that the epitope recognized by the BPA antibody is formed by the phenol and methyl groups of BPA [Figure 1]a. Additional ELISA assays were carried out with 10 pg/ml –1 μg/ml of BPA using a plate coated with BPA antibody [Figure 1]b. In addition, electrophoresis and immunoblot analysis performed with BSA or BSA-BPA conjugates and goat anti-BPA IgG demonstrated that the antibody identified BPA and did not cross-react with BSA [Figure 2].
|Figure 1: Cross-reactivity of anti-BPA IgG with BPF, BPS and resveratrol. ELISA carried out with the range of 10 pg/ml – 1 μg/ml. The cross-reactivity tests with BPF (no methyl group), BPS (methyl group substituted with a sulfone) and resveratrol were calculated using the concentration required to displace 50% of the BPA-HRP signal in BPA ELISA. (a) Molecular structures of BPA, BPS, BPF and resveratrol (b) Standard curve for the BPA immunoassay. HRP: Horseradish peroxidase, BPA: Bisphenol A, ELISA: Enzyme-linked immunosorbent assay, BPF: Bisphenol F, BPS: Bisphenol S|
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|Figure 2: BPA was conjugated to BSA for specificity of the BPA antibody to identify BPA. (a) Amido black staining. Lane 1, 2 μg BSA. Lane 2, 0.2 μg BSA conjugated to BPA. (b) Immunoblot using goat anti-BPA antibody raised against a BPA-keyhole limpet hemocyanin (KLH) antigen. Lane 1, 2 μg BSA. Lane 2, 0.2 μg BSA conjugated to BPA (arrow indicates to the correct molecular weight of monomeric BPA-BSA conjugate). BSA polymers were formed during BPA conjugation to BSA. BPA: Bisphenol A, BSA: Bovine serum albumin|
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Measurement of bisphenol A in commercial soups
Liquid obtained from canned soup was spiked with 5 μl DMF with or without 10 ng BPA. The competitive ELISA demonstrated that approximately 95% of the added BPA was recovered [Table 1]. As shown in [Figure 3]a, there was no difference on BPA levels in different types of soup from the same company in USA (Country #1). The mean levels of BPA in soup supernatants varied between 8 to 10.5 ng/ml. Also there was no difference in the BPA levels of canned vegetable soup from different companies in USA. A comparison of BPA levels in canned vegetable soup with canned vegetable mix produced in USA was also made. Levels of BPA in the supernatant of canned vegetable mix was 200-fold lower compared to the canned vegetable soup from two different US companies. There was high variability of BPA levels among companies from different countries varying from 7 to ~200 ng/ml [Figure 3]b. Canned soup produced in the USA (Country #1) had lower BPA levels as compared to the canned soup produced by two different companies from two of the Asian countries (Countries #3 and #4). Canned food from Country #4 had the highest BPA levels of BPA in supernatants of canned soups [Figure 3]b. BPA levels in products from Counties #2 and #4 were independent of the company while there was a difference of almost 90% in levels of BPA in Country #3 depending on the brand name [Figure 3]b.
|Table 1: Percentage Recovery of BPA in supernatant of canned soup spiked with BPA. DMF (5 μl), with or without 10 ng BPA, was added to 1 ml of canned soup supernatant. The supernatant was diluted 10-fold and competitive ELISA was carried out using BPA-conjugated HRP. BPA: bisphenol A, DMF: Dimethylformamide, SD: Standard deviation, ELISA: Enzyme-linked immunosorbent assay, HRP: Horseradish peroxidase|
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|Figure 3: BPA levels of supernatants of canned foods in USA (Country #1) from different countries. (a) Levels of BPA in the supernatant were measured using a competitive BPA ELISA in different types of soup from the same company of Country #1. (b) Levels of BPA in the supernatants of canned food from companies of different countries were measured using a competitive BPA ELISA from companies of different countries. *P < 0.05 from product within countries, #P < 0.05 from average of Country #1. BPA: bisphenol A, ELISA: enzyme-linked immunosorbent assay|
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Comparison of bisphenol A level assessed by enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry
BPA levels in supernatants of canned foods from Country #1 (Companies A and B) were similar and their BPA levels as assessed by ELISA were confirmed by LC/MS/MS analysis. Both the pattern and absolute concentration of BPA in supernatants from these two companies as determined by LC/MS/MS were in agreement with the ELISA results [Table 2].
|Table 2: Levels of bisphenol A in canned soups measured by enzyme-linked immunosorbent assay and liquid chromatography-mass-spectrometry/mass-spectrometry|
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| Discussion|| |
The presence of BPA in blood and urine is associated with infertility as well as the incidence of a large number of diseases that are leading causes of death worldwide.,,, Despite of increased regulations to control the use of BPA in food packaging, the human population is continuously exposed to BPA., Thus, it is highly important for public health that easy and rapid strategies for monitoring BPA levels in food and beverages are developed. For the first time, this study presents evidence to support the efficacy of a BPA ELISA assay sensitive enough for the measurement of BPA in supernatants of canned food, which is a simple and fast technique that does not require extraction steps.
Canned foods are one of the main sources of dietary BPA due to the leaching of BPA from the epoxy resin lining of cans. FDA laboratories measured BPA levels in canned food solids from different companies in the USA by LC/MS/MS after acetonitrile extraction and levels ranged from 13 to 110 ng/g. A subsequent 2 × 2 crossover study analyzing urinary BPA levels in 75 voluntary subjects using LC/MS/MS revealed that a 12-ounce serving of canned soup consumption for 5 days increased the urinary BPA level of the individuals by ~12-fold. In that study, the group that consumed a 12-ounce serving/day of canned soups for 5 days had urine levels of 20.8 μg/ml BPA whereas the control group that consumed equal amounts of fresh soups had urine levels of only 1.1 ng/ml BPA. Moreover, analyses conducted by the National Health and Nutrition Examination Survey after evaluating 7,669 people between 2003 and 2008 confirmed that urinary BPA concentrations increased with increased frequency of consumption of canned foods, and specifically with the ingestion of canned vegetable, fruit, pasta, and soup. In agreement, a great number of studies have demonstrated a high level of BPA in different types of canned food and beverages even after the legislation limits the use of BPA in food packaging., The complexity, cost, sensitivity and requirement for extraction steps for LC/MS/MS methodology used to determine BPA levels might be one of the reasons for limiting the constant assessment of BPA levels in food and beverages. Therefore, new strategies have been developed to improve the simplicity and expense of BPA measurements., Moreno et al. developed an antibody-based immunoassay to simplify BPA analyses in supernatants of canned food. Despite their very original and innovative study, the limit of detection of the immunoassays developed by Moreno and colleagues could not detect very low levels of BPA in supernatants of canned food without a sample concentration step. Here, we present data showing that the use of an ultrasensitive ELISA assay for BPA measurement in food supernatants without a concentration step is as suitable as traditional analytical methods. The antibody used to capture BPA has a high specificity and the limit of detection of the ELISA is <10 pg/ml, which is lower than traditional techniques used to measured BPA (>1 ng/ml) and other BPA ELISA.,
BPA in liquid supernatants of canned foods mostly originates from the leaching of BPA from the lining material used to coat the inside of the cans. With increase in regulations to limit BPA use in a few developed countries such as the USA and Canada, the canning industry started to invest in the fabrication of BPA-free can linings. However, using LC/MS/MS, researchers from the FDA detected BPA in 63 of 105 canned foods, showing that the assessment of BPA is very important because its levels varied among canned food from different companies. In this study, we found an almost 200-fold difference in BPA levels when comparing canned vegetable mix supernatant with canned vegetable soup. In addition, we demonstrated that BPA varies between countries that package similar food. Difference in BPA concentration might be a result of legislation that controls the use of BPA by the packaging industry. It should be mentioned that the country of origin of epoxy resin used to line steel cans may be different from the country where the food is ultimately canned.
Importantly, the pattern and absolute concentrations of BPA in canned food as determined by ELISA were similar to the traditional LC/MS/MS methodology used to assess BPA in canned food. This result confirmed that the BPA ELISA is suitable for the analyses of BPA in supernatants of canned food and beverages. The agreement between ELISA and LC/MS/MS analyses was also found when levels of BPA were quantified in sewage water from different communities of Detroit, Michigan. It should be noted that the assessment of BPA by ELISA does not require extraction steps, which makes this technique faster, easier and more suitable to large-scale analyses of BPA in canned food.
The authors would like to thank the Lumigen Instrument Center, Wayne State University for the use of Shimadzu Nextera X2 UPLC/MS/MS which was purchased through support of a Shimadzu grant and the technical assistance provided by Johnna Birbeck and Nicole Lenca.
Financial support and sponsorship
Conflict of interest
Klaus Friedrich and So Hee Kim declare that they have no competing interests. Hyesook Kim is the president of Detroit R&D, Inc. and has a commercial interest. David Kaplan, Aby Joiakim, Julia Matzenbacher Santos and David A. Putt also work for Detroit R&D, Inc.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]