Polyphenol exposure of mothers and infants assessed by LC-MS/MS based biomonitoring in breast milk.

Exposure to polyphenols is relevant throughout critical windows of infant development, including the breastfeeding phase. However, the quantitative assessment of polyphenols in human breast milk has received limited attention so far, though polyphenols may positively influence infant health. Therefore, a targeted LC-MS/MS assay was developed to investigate 86 analytes representing different polyphenol classes in human breast milk. The sample preparation consisted of liquid extraction, salting out, freeze-out, and a dilution step. Overall, nearly 70% of the chemically diverse polyphenols fulfilled all strict validation criteria for full quantitative assessment. The remaining analytes did not fulfill all criteria at every concentration level, but can still provide useful semi-quantitative insights into nutritional and biomedical research questions. The limits of detection for all analyzed polyphenols were in the range of 0.0041-87 ng*mL-1, with a median of 0.17 ng*mL-1. Moreover, the mean recovery was determined to be 82% and the mean signal suppression and enhancement effect was 117%. The developed assay was applied in a proof-of-principle study to investigate polyphenols in breast milk samples provided by twelve Nigerian mothers at three distinct time points post-delivery. In total, 50 polyphenol analytes were detected with almost half being phenolic acids. Phase II metabolites, including genistein-7-ß-D-glucuronide, genistein-7-sulfate, and daidzein-7-ß-D-glucuronide, were also detected in several samples. In conclusion, the developed method was demonstrated to be fit-for-purpose to simultaneously (semi-) quantify a wide variety of polyphenols in breast milk. It also demonstrated that various polyphenols including their biotransformation products were present in breast milk and therefore likely transferred to infants where they might impact microbiome development and infant health.

Quantification and stability assessment of 7,9-di­tert­butyl­1-oxaspiro(4,5)deca-6,9-diene-2,8­dione leaching from cross-linked polyethylene pipes using gas and liquid chromatography.

This study assesses the formation and stability of the water contaminant 7,9-di­tert­butyl­1-oxaspiro(4,5)deca-6,9-diene-2,8­dione ([1]) which repeatedly occurs in the migration waters of cross-linked polyethylene (PE-X) pipes. In aqueous solution [1] is partially transformed to 3-(3,5-di­tert­butyl­1­hydroxy-4-oxo-2,5-cyclohexadien-1-yl)propionic acid ([2]). For a better understanding of the formation of [1] and its transformation into [2] an analytical method was established to allow the analysis of both species separately. Because of thermal instability [2] cannot be detected with GC-MS. Therefore, two methods were validated for a reliable and reproducible quantification: GC-MS for [1] and HPLC-MS/MS for both [1] and [2]. Comparative measurements of migration waters from PE-X pipes using GC-MS and HPLC-MS/MS methods showed that the concentrations of [1] detected with GC-MS corresponds to the sum of [1] and [2] measured with HPLC-MS/MS. In the migration waters [1] was detected in higher concentrations than [2]. The highest concentrations of [1], detected with GC-MS, were > 300 µg/L. The longer the materials are stored without contact with water, the more [1] is measured in the migration waters. Most of the previous values reported in the literature for [1] were based on semi-quantification. Hence, we compared results of the semi-quantitative determination according to EN 15768 with those of a quantitative method with a standard. The results gained with the semi-quantitative method represent less than 50% of the quantified values for the amount leaching from the pipes, which means that the semi-quantification method according to EN 15768 leads to a significant underestimation of [1]. Finally, stability assessment showed that [1] developed an equilibrium with [2] under acidic conditions, whereas it will completely be transferred to [2] at pH 10. At pH 7, it takes more than 50 days for [1] to reach an equilibrium with [2]. However, at increasing the temperature to 60 °C, [1] will be rapidly transformed into [2]. Besides [1] and [2], other currently unknown degradation products are formed. As there is no comprehensive toxicological assessment for both substances available today, our findings underline the need for regulatory consequences.

Exposomic Biomonitoring of Polyphenols by Non-Targeted Analysis and Suspect Screening.

Polyphenols, prevalent in plants and fungi, are investigated intensively in nutritional and clinical settings because of their beneficial bioactive properties. Due to their complexity, analysis with untargeted approaches is favorable, which typically use high-resolution mass spectrometry (HRMS) rather than low-resolution mass spectrometry (LRMS). Here, the advantages of HRMS were evaluated by thoroughly testing untargeted techniques and available online resources. By applying data-dependent acquisition on real-life urine samples, 27 features were annotated with spectral libraries, 88 with in silico fragmentation, and 113 by MS1 matching with PhytoHub, an online database containing >2000 polyphenols. Moreover, other exogenous and endogenous molecules were screened to measure chemical exposure and potential metabolic effects using the Exposome-Explorer database, further annotating 144 features. Additional polyphenol-related features were explored using various non-targeted analysis techniques including MassQL for glucuronide and sulfate neutral losses, and MetaboAnalyst for statistical analysis. As HRMS typically suffers a sensitivity loss compared to state-of-the-art LRMS used in targeted workflows, the gap between the two instrumental approaches was quantified in three spiked human matrices (urine, serum, plasma) as well as real-life urine samples. Both instruments showed feasible sensitivity, with median limits of detection in the spiked samples being 10-18 ng/mL for HRMS and 4.8-5.8 ng/mL for LRMS. The results demonstrate that, despite its intrinsic limitations, HRMS can readily be used for comprehensively investigating human polyphenol exposure. In the future, this work is expected to allow for linking human health effects with exposure patterns and toxicological mixture effects with other xenobiotics.

Differential response of human glioblastoma cell lines to combined camptothecin and ionizing radiation treatment.

In order to enhance the cytotoxicity of radiation, camptothecin (CPT), an inhibitor of DNA topoisomerase I, was added to the cultured glioma cell lines before irradiation (IR). Radiation responses of five glioblastoma cell lines (U87-MG, U373-MG, GHE, GaMG and SNB-19) treated with CPT were analyzed in terms of cell and colony counts, cell cycle progression, expression of histone gamma H2AX, DNA repair protein Rad50, survivin, cleaved caspase 3, p53 and of topoisomerase I. CPT enhanced the radiotoxicity in U87-MG and SNB-19 cell lines if cell and colony counts were used as the end-points. In contrast, pre-treatment with CPT of U373-MG, GHE and GaMG cell lines did not enhance cytotoxicity of IR in terms of cell and colony counts but accelerated DNA damage repair assessed by Rad50 foci. CPT treated glioma cells revealed at least two subpopulations with respect to the expression of histone gamma H2AX, a marker of DNA double-strand breaks. The cell lines tested also differed in the expression of survivin, cleaved caspase 3, p53 and of topoisomerase I. The failure of CPT to enhance the radiotoxicity of glioma U373-MG, GHE and GaMG cell lines in terms of cell and colony counts was found to correlate with accelerated DNA damage repair, and with low expression of topoisomerase I, a target of CPT.