Assessing the stability of polyphenol content in red rooibos herbal tea using traditional methods and high-resolution mass spectrometry: Implications for studying dietary interventions in preclinical rodent studies.

Preclinical rodent models are used to examine the relationship between tea consumption and bone health, where tea is available for rodents and typically replaced weekly. However, the extent to which the tea polyphenols change over time remains uncertain, despite its importance in preparing tea during preclinical rodent trials. Using an untargeted molecular approach, we applied a liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOFMS) system to assess the molecular profile of red rooibos teas throughout a 6-day aging period. We found a significant, 3-fold decrease of polyphenols involved in bone metabolism, including m-coumaric acid, catechin derivatives and courmaroyl tartaric acid over 6 days, likely due to photochemical decomposition and autooxidation within tea extracts. Using a novel untargeted workflow for polyphenol characterization, our findings revealed the complexity of polyphenols in red rooibos teas that can inform the evidence-based decisions of how often to change teas during in vivo rodent trials.

Phytoplankton metabolite profiles from two Lake Ontario Areas of Concern reveal differences associated with taxonomic community composition.

Water quality and phytoplankton community composition are important factors that can indicate freshwater ecosystem health. We combined water quality, phytoplankton community, and metabolomic data from algae and water sampled from two embayments in Lake Ontario, Hamilton Harbour and the Bay of Quinte, over ten weeks from August to October in the year 2020. Metabolomics was performed using liquid chromatography tandem mass spectroscopy (LC-MS/MS) to identify changes in intracellular metabolites within algae communities over time, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to characterize putative isomers of extracellular metabolites at sub-ppb mass accuracy. Results from this study indicate that Hamilton Harbour and the Bay of Quinte are two very different ecosystems with respect to water quality, phytoplankton metabolites, and phytoplankton community composition. Community composition is strongly driven by conductivity and nitrates in Hamilton Harbour, while the opposite is true in the Bay of Quinte. Metabolites including α-aminobutyric acid and glycine were found in larger abundance within algal communities at both locations, while taurine was more predominant in algal communities from the Bay of Quinte. These metabolic profiles could reflect the different communities of phytoplankton, and be alternative indicators of algal bloom growth.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Mercury methylation and methylmercury demethylation in boreal lake sediment with legacy sulphate pollution.

Sulphate and dissolved organic matter (DOM) in freshwater systems may regulate the formation of methylmercury (MeHg), a potent neurotoxin that biomagnifies in aquatic ecosystems. While many boreal lakes continue to recover from decades of elevated atmospheric sulphate deposition, little research has examined whether historically high sulphate concentrations can result in persistently elevated MeHg production and accumulation in aquatic systems. This study used sediment from a historically sulphate-impacted lake and an adjacent reference lake in northwestern Ontario, Canada to investigate the legacy effects of sulphate pollution, as well as the effects of newly added sulphate, natural organic matter (NOM) of varying sulphur content and a sulphate reducing bacteria (SRB) inhibitor on enhancing or inhibiting the Hg methylation and demethylation activity (Kmeth and Kdemeth) in the sediment. We found that Kmeth and MeHg concentrations in sulphate-impacted lake sediment were significantly greater than in reference lake sediment. Further adding sulphate or NOM with different sulphur content to sediment of both lakes did not significantly change Kmeth. The addition of a SRB inhibitor resulted in lower Kmeth only in sulphate-impacted sediment, but methylation was not entirely depressed. Methylmercury demethylation potentials in sediment were consistent across lakes and experimental treatments, except for some impacts related to SRB inhibitor additions in the reference lake sediment. Overall, a broader community of microbes beyond SRB may be methylating Hg and demethylating MeHg in this system. This study reveals that legacies of sulphate pollution in boreal lakes may persist for decades in stimulating elevated Hg methylation in sediment.

An estimation of sulfur concentrations released by three algae (Chlorella vulgaris, Chlamydomonas reinhardtii, Scenedesmus obliquus) in response to variable growth photoperiods.

In this study, we estimated extracellular concentrations of algal-derived sulfur species in response to changing photoperiods. Cultures from three algal species (Chlorella vulgaris, Chlamydomonas reinhardtii, Scenedesmus obliquus) were subjected to five different light:dark cycles (12:12, 14:10, 16:8, 18:6, 20:4 h) for a period of 3 days. Sulfur compounds including total reactive thiol concentrations, electroactive reduced sulfur species (RSS), and thiol isomers were measured using qBBr fluorescence, differential pulse cathodic stripping voltammetry (DP-CSV), and high-resolution mass spectrometry (HRMS), respectively. Total reactive thiol concentrations were greater in Scenedesmus than in Chlamydomonas and Chlorella at low light regimes (i.e., 12:12 h) whereas Chlamydomonas produced more RSS than the other two species (p < 0.05) at any light regime. Scenedesmus was the only responsive species to produce maximal electroactive RSS, and HRMS equivalent thiol compounds under low light regime, congruent with previous studies. Principal component analysis revealed relationships between qBBr-equivalent thiol and GSH-equivalent RSS concentrations for Scenedesmus and Chlamydomonas (p < 0.05) suggesting that thiols were the dominant species in the pool of electroactive RSS for these two algal species. Overall, these results showed that the light growth conditions greatly influenced the production of S-rich compounds by algae, affecting the complexation of metals such as mercury and cadmium, especially during planktonic blooms.

Aerobic and Anaerobic Bacterial Mercury Uptake is Driven by Algal Organic Matter Composition and Molecular Weight.

The biological mobilization of mercury (Hg) into microbes capable of Hg methylation is one of the limiting steps in the formation of the neurotoxin methylmercury (MeHg). Although algal dissolved organic matter (DOM) has been associated with increased MeHg production, the relationship between bacterial Hg uptake and algal DOM remains unexplored. In this study, we aimed to address how the quantity and quality of DOM, freshly harvested from several algae, affected the bacterial uptake of Hg with the use of a biosensor capable of functioning both aerobically and anaerobically. We combined biosensor measurements with high-resolution mass spectrometry and field-flow fractionation to elucidate how DOM composition and molecular weight influenced microbial Hg uptake. We showed that freshly harvested DOM from Chlorophyte and Euglena mutabilis strongly inhibited aerobic and anaerobic Hg uptake, whereas DOM harvested from Euglena gracilis did not exhibit this same pronounced effect. Once fractionated, we found that amino acids and polyamines, most abundant in Euglena gracilis DOM, were positively correlated to increase Hg uptake, suggesting that these molecules are potentially underappreciated ligands affecting Hg bioavailability. As water quality is affected by eutrophication, algal community assemblages will change, leading to variations in the nature of autochthonous DOM released in aquatic systems. Our results highlight that variations in the emergent properties of DOM originating from varying algal species can have a profound effect on bacterial Hg uptake and thus methylation.

Influence of dissolved organic matter on dissolved vanadium speciation in the Churchill River estuary (Manitoba, Canada).

Diffusive gradients in thin films (DGT) devices were used to investigate the temporal and spatial changes in vanadium (V) speciation in the Churchill estuary system (Manitoba). Thirty-six DGT sets and 95 discrete water samples were collected at 8 river and 3 estuary sites during spring freshet and summer base flow. Dissolved V concentration in the Churchill River at summer base flow was approximately 5 times higher than those during the spring high flow (27.3 ± 18.9 nM vs 4.8 ± 3.5 nM). DGT-labile V showed an opposite trend with greater values found during the spring high flow (2.6 ± 1.8 nM vs 1.4 ± 0.3 nM). Parallel factor analysis (PARAFAC) conducted on 95 excitation-emission matrix spectra validated four humic-like (C1C4) and one protein-like (C5) fluorescent components. Significant positive relationship was found between protein-like DOM and DGT-labile V (r = 0.53, p < 0.05), indicating that protein-like DOM possibly affected the DGT-labile V concentration in Churchill River. Sediment leachates were enriched in DGT-labile V and protein-like DOM, which can be readily released when river sediment began to thaw during spring freshet.

Molecular characterization of phytoplankton dissolved organic matter (DOM) and sulfur components using high resolution Orbitrap mass spectrometry.

Orbitrap high resolution mass spectrometry (HRMS) with electrospray ionization in both positive and negative polarity was conducted on Suwannee River fulvic acid (SRFA), Pony Lake fulvic acid (PLFA) standards, and dissolved organic matter (DOM) released by freshwater phytoplankton (Scenedesmus obliquus, Euglena mutabilis, and Euglena gracilis). Three-dimensional van Krevelen diagrams expressing various oxygenation states of sulfur molecules and abundance plots of sulfur-containing species were constructed. Orbitrap HRMS analysis of SRFA found a high density of peaks in the lignin region (77 %) and low density of protein material (6.53 %), whereas for PLFA, 25 % of the total peaks were lignin related compared to 56 % of peaks in protein regions, comparable with other HRMS studies. Phytoplankton-derived DOM of S. obliquus, E. mutabilis, and E. gracilis was dominated by protein molecules at respective percentages of 36, 46, and 49 %, and is consistent with previous experiments examining phytoplankton-derived DOM composition. The normalized percentage of SO-containing compounds was determined among the three phytoplankton to be 56 % for Scenedesmus, 54 % for E. mutabilis, and 47 % for E. gracilis, suggesting variation between sulfur content in phytoplankton-derived DOM and differences in metal binding capacities. These results suggest the level of resolution by Orbitrap mass spectrometry is sufficient for preliminary characterization of phytoplankton DOM at an affordable cost relative to other HRMS techniques.

Examining concentrations and molecular weights of thiols in microorganism cultures and in Churchill River (Manitoba) using a fluorescent-labeling method coupled to asymmetrical flow field-flow fractionation.

In this study, molecular weights of thiols from four laboratory cultures (Scenedesmus obliquus, Chlorella vulgaris, Euglena gracilis, and Attheya septentrionalis) and the Churchill River (Manitoba) were assessed using a fluorescent-labeling method such as monobromotrimethylammoniobimane (qBBr) and asymmetrical flow field-flow fractionation (AF4) coupled to a fluorescence detector. Concentrations of thiols in extracellular fractions ranged from 6.39 ± 3.39 to 39.2 ± 7.43 µmol g(-1), and intracellular concentrations ranged from 11.5 ± 4.52 to 41.0 ± 4.1 µmol g(-1). In addition, molecular weights (MW) of intracellular thiol ranged from 493 ± 24 to 946 ± 12 Da whereas extracellular thiol MWs varied from 443 ± 36 to 810 ± 174 Da. The novel method of combining AF4 to an on-line fluorometer and the incorporation of the thiol tag provided information regarding thiol concentration and composition of controlled and natural systems. Furthermore, the proposed methods allow for the simultaneous measurement of thiol and DOM MWs produced by microorganisms. By assessing characteristics of naturally produced thiols and lab-grown thiols, information regarding heavy metal complexation can be determined.