A round robin approach to the analysis of bisphenol a (BPA) in human blood samples

BACKGROUND: Human exposure to bisphenol A (BPA) is ubiquitous, yet there are concerns about whether BPA can be measured in human blood. This Round Robin was designed to address this concern through three goals: 1) to identify collection materials, reagents and detection apparatuses that do not contribute BPA to serum; 2) to identify sensitive and precise methods to accurately measure unconjugated BPA (uBPA) and BPA-glucuronide (BPA-G), a metabolite, in serum; and 3) to evaluate whether inadvertent hydrolysis of BPA-G occurs during sample handling and processing. METHODS: Four laboratories participated in this Round Robin. Laboratories screened materials to identify BPA contamination in collection and analysis materials. Serum was spiked with concentrations of uBPA and/or BPA-G ranging from 0.09-19.5 (uBPA) and 0.5-32 (BPA-G) ng/mL. Additional samples were preserved unspiked as ‘environmental’ samples. Blinded samples were provided to laboratories that used LC/MSMS to simultaneously quantify uBPA and BPA-G. To determine whether inadvertent hydrolysis of BPA metabolites occurred, samples spiked with only BPA-G were analyzed for the presence of uBPA. Finally, three laboratories compared direct and indirect methods of quantifying BPA-G. RESULTS: We identified collection materials and reagents that did not introduce BPA contamination. In the blinded spiked sample analysis, all laboratories were able to distinguish low from high values of uBPA and BPA-G, for the whole spiked sample range and for those samples spiked with the three lowest concentrations (0.5-3.1 ng/ml). By completion of the Round Robin, three laboratories had verified methods for the analysis of uBPA and two verified for the analysis of BPA-G (verification determined by: 4 of 5 samples within 20% of spiked concentrations). In the analysis of BPA-G only spiked samples, all laboratories reported BPA-G was the majority of BPA detected (92.2 – 100%). Finally, laboratories were more likely to be verified using direct methods than indirect ones using enzymatic hydrolysis. CONCLUSIONS: Sensitive and accurate methods for the direct quantification of uBPA and BPA-G were developed in multiple laboratories and can be used for the analysis of human serum samples. BPA contamination can be controlled during sample collection and inadvertent hydrolysis of BPA conjugates can be avoided during sample handling

Enhancement of Lipopolysaccharide-Induced Neutrophil Oxygen Radical Production by Tumor Necrosis Factor Alpha

Although tissues become exposed to both exogenous and endogenous cell-activating mediators during infection, there is little appreciation of the effects of subjecting cells to multiple mediators. We examined the hypothesis that the response of neutrophils to bacterial lipopolysaccharide (LPS) is significantly altered in the presence of the endogenous mediator tumor necrosis factor alpha (TNF). The data showed that human neutrophils pretreated with TNF for 10 to 30 min, displayed significantly enhanced superoxide production in response to LPS (from either Escherichia coli K-235 or E. coli 0127:B8), measured as lucigenin-dependent chemiluminescence (CL), seen as an increase in the initial peak rate as well as the total CL accumulated over the incubation period. TNF amplified the response to LPS at 1 to 100 U of TNF/10(6) neutrophils and was able to enhance the response to a wide range of concentrations of LPS (0.01 to 1,000 ng/ml). The TNF-induced increase in the LPS response was paralleled by an increase in LPS binding to the neutrophils, which could be abrogated by an anti-CD14 monoclonal antibody. The results demonstrate that TNF significantly increases the LPS-induced release of oxygen radicals in neutrophils through the upregulation of cell surface CD14