A miniaturized sample preparation method for routine elemental determination in whole blood using volumetric absorptive micro-sampling by ICP-QQQ.

Volumetric absorptive micro-sampling (VAMS) has emerged as a simple and safe tool for collecting and storing blood samples in clinical and bioanalytical fields. This study presents a novel method for determining essential and non-essential trace elements (As, Be, Cd, Cs, Cu, Fe, Mg, P, Pb, S, Sb, Se, Tl, V, U) in VAMS-collected blood samples using microwave-assisted digestion with diluted acid as sample preparation method and an inductively coupled plasma triple quadrupole mass spectrometry (ICP-QQQ) as determination technique. While certain elements posed challenges due to VAMS tip background issues (Al, Ti, Cr, Mn, Co, Ni, Sn, Mo, Ba), the method demonstrated high precision and accuracy for the targeted analytes. It was demonstrated that 4.5 mol L-1 HNO3 plus 100 µL H2O2 30% (w/w) was suitable for an efficiency of digestion for further elemental determination using micro-analysis (spending less than 300 µL analytical solution) by ICP-QQQ, given that the residual carbon content (RCC) after the digestion procedure was lower than 5%. All the results higher than limit of quantification (LOQ) were in agreement with reference values for all analytes. Accuracy was assessed through reference material analysis and recovery tests using spiked samples. Moreover, suitable agreements (p > 0.05) between this method (VAMS-M) and the comparative method (liquid sampling method) were obtained for all analytes >LOQ. Furthermore, all results >LOQ showed good precision according to precision requirements (Horwitz equation). In this way, with the use of dilute acid, low dilution factor (30-fold), and excellent digestion efficiency (>95%), the proposed method was able to achieve an excellent detection limit, precision, and accuracy for 15 elements: As, Be, Cd, Cs, Cu, Fe, Mg, P, Pb, S, Sb, Se, Tl, V, and U using ICP-MS/MS, without the need for matrix-matched calibration curves. This research showcases an innovative analytical approach using VAMS for blood samples, offering biosafety, practicality, sensitivity, versatility, and robustness. This method contributes to the advancement of trace element analysis in biomedical research and clinical applications.

Feasibility study for mercury remediation by selenium competition in Pleurotus mushrooms.

Mushrooms may incorporate significant levels of Hg making its consumption harmful to human health. Mercury remediation induced by Se competition in edible mushrooms represents a valuable alternative since Se plays effective roles against Hg uptake, accumulation, and toxicity. In this way, Pleurotus ostreatus and Pleurotus djamor were cultivated on Hg-contaminated substrate simultaneously supplemented with Se(IV) or Se(VI) under different dosages in this study. The protective role of Se was assessed taking into account morphological characteristics and Hg and Se total concentrations determined by ICP-MS, as well as proteins and protein-bound Hg and Se distribution by SEC-UV-ICP-MS, and Hg speciation studies (Hg(II) and MeHg) by HPLC-ICP-MS. Both Se(IV) and Se(VI) supplementation were able to recover the morphology mainly of Hg-contaminated Pleurotus ostreatus. The mitigation effects induced by Se(IV) stood out more than Se(VI) in terms of Hg incorporation, decreasing the total Hg concentration up to 96 %. Also, it was found that supplementation mainly with Se(IV) reduced the fraction of Hg bound to medium molecular weight compounds (17-44 kDa) up to 80 %. Finally, it was shown a Se-induced inhibitory effect on Hg methylation, decreasing MeHg species content in mushrooms exposed to Se(IV) (51.2 µg g-1) up to 100 %.

The protective role of selenium against uptake and accumulation of cadmium and lead in white oyster (Pleurotus ostreatus) and pink oyster (Pleurotus djamor) mushrooms.

Mushrooms are bioaccumulators and have been used to produce Se-enriched foods. However, these fungi can also bioaccumulate potentially toxic metals, producing food dangerous to human health. It is known that co-exposure to Se plays a protective role against metal accumulation and toxicity in some organisms due to its antioxidant properties. Thus, this study aimed to evaluate the protective effect of Se(IV) and Se(VI) on elemental uptake and accumulation as well as proteins and protein-bound Se, Cd, and Pb distribution in Pleurotus mushrooms. Pink oyster and white oyster mushrooms showed high ability to bioaccumulate Se (19-205 µg g-1), Cd (4.5 to 18.8 µg g-1), and Pb (1.6 to 7.0 µg g-1). Growth substrate supplementation with Se(IV) or Se(VI) decreased the Cd total concentration in mushrooms by 4 to 89%, while Se(VI) increased the Pb total concentration by 9% to 187%, compared to growth in absence of Se. It was found that despite molecular weights distributions of mushrooms grown on Se(IV) and Se(VI)-supplemented substrates being similar, Se(VI) supplementation favoured Se interaction with proteins of medium molecular weight (17-44 kDa), when compared to supplementation with Se(IV). Therefore, we propose the supplementation of growth substrates with Se(VI) to reduce eventual Cd accumulation and produce Se-enriched oyster mushrooms.

Effects of Se(IV) or Se(VI) enrichment on proteins and protein-bound Se distribution and Se bioaccessibility in oyster mushrooms.

A successful mushroom enrichment process must produce foods that have compounds potentially absorbed by the human body. In this study, Pleurotus ostreatus and Pleurotus djamor mushrooms were grown on organic substrate supplemented with different Se(IV) and Se(VI) concentrations, and evaluated in the following features: Fruiting bodies morphology; Se uptake and accumulation; Distribution of proteins and protein-bound Se; Se species identification on enzymatic extracts; Se bioaccessibility; and Distribution of bioaccessible protein-bound Se. Pleurotus djamor grown on Se(IV)-supplemented substrate showed the greatest potential to uptake and accumulate Se. For Se species screening, selenomethionine was identified in white oyster mushroom, while selenomethionine, selenocystine, and Se-methylselenocysteine in pink oyster mushrooms. In soluble fractions from in vitro gastrointestinal digestion assays, Se showed high bioaccessibility (>94%). Lastly, bioaccessible Se species were found to be mainly associated to LMW (<17 kDa) in Pleurotus ostreatus (74%) and Pleurotus djamor (68%) grown on Se(IV)-supplemented substrates.

Effect of Fusarium oxysporum f. sp. lycopersici on the degradation of humic acid associated with Cu, Pb, and Ni: an in vitro study.

The intent of this work was to gain further insight on the fungus-assisted degradation/solubilization of humic acid and the related changes in metal-binding profiles. In the experimental design, Aldrich reagent humic acid (HA) or HA enriched with Cu, Pb, and Ni (HA(Me)) was added to Fusarium oxysporum f. sp. lycopersici cultures in vitro. The cultures were supplied by different carbon- and nitrogen-containing nutrients (glucose, Glc, or glutamate, Glu and ammonium, NH4+, or nitrate, NO3-, ions, respectively) in order to examine their possible effect on HA and HA(Me) decomposition. During the first 48 h of fungus growth, gradual acidification to pH 2 was observed in medium containing Glc + NH4+, while for other cultures, alkalinization to pH 9 occurred and then, the above conditions were stable up to at least 200 h. Size exclusion chromatography (SEC) with UV/Vis detection showed progressive degradation and solubilization of both HA and HA(Me) with the increasing time of fungus growth. However, the molecular mass distributions of HA-related soluble species were different in the presence of metals (HA(Me)) as referred to HA and were also influenced by the composition of growth medium. The solubilization of Pb, Cu, and Ni and their association with HA molecular mass fractions were studied using inductively coupled plasma mass spectrometry (ICP-MS) detection. Under acidic conditions, relatively high concentrations of low-molecular-mass metallic species were found in culture supernatants, while in alkaline media, metal solubilization was generally poorer. In contrast to low pH culture, SEC-ICP-MS results obtained in alkaline supernatants indicated metal binding to degradation products of humic substances of MM > 5 kDa. In summary, the results of this study suggest that fungus-assisted degradation of HA and HA(Me) might be controlled using appropriate N- and C- sources required for fungus growth, which in turn would affect molecular mass distribution of soluble metallic species thus potentially influencing their actual bioaccessibility.

Phosphorus and osmium as elemental tags for the determination of global DNA methylation--a novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies.

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2'-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on (31)P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K(2)OsO(4)) in the presence of strong oxidant (K(3)Fe(CN)(6)) and N,N,N',N'-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography-ICP-MS for specific detection at (31)P and (189)Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC-DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.

Effect of Fusarium oxysporum f. sp. lycopersici on the soil-to-root translocation of heavy metals in tomato plants susceptible and resistant to the fungus.

The purpose of this work was to gain an insight on the potential role of the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici in the translocation of metals and metalloids from soil to plant roots in tomato (Lycopersicum esculentum). Two varieties of tomato (one susceptible and another resistant to infection by Fusarium oxysporum f. sp. lycopersici) were challenged with the fungus for different periods of time, and several elements (V, Cr, Mn, Co, Cu, Zn, As, Se, Mo, Ag, Cd, Pb) were determined in roots and in soil substrate. Additionally, phenolic plant products were also analyzed for the evaluation of the plant response to biotic stress. In order to obtain representative results for plants cultivated in noncontaminated environments, the infected and control plants were grown in commercial soil with natural, relatively low metal concentrations, partly associated with humic substances. Using such an experimental design, a specific role of the fungus could be observed, while possible effects of plant exposure to elevated concentrations of heavy metals were avoided. In the infected plants of two varieties, the root concentrations of several metals/metalloids were increased compared to control plants; however, the results obtained for elements and for phenolic compounds were significantly different in the two plant varieties. It is proposed that both Lycopersicum esculentum colonization by Fusarium oxysporum f. sp. lycopersici and the increase of metal bioavailability due to fungus-assisted solubilization of soil humic substances contribute to element traffic from soil to roots in tomato plant.