Search Results (280)

Functional foods like flax (Linum usitatissimum L.) are rich sources of specialized metabolites that contribute to their nutritional and health-promoting properties. Understanding the biosynthesis of these compounds is essential for improving their quality and potential applications. However, dissecting complex metabolic networks in plants remains challenging due to the dynamic nature and interconnectedness of biosynthetic pathways. In this study, we present a synergistic approach combining stable isotopic labeling (SIL), Candidate Substrate–Product Pair (CSPP) networks, and a time-course study with high temporal resolution to reveal the biosynthetic fluxes shaping phenylpropanoid metabolism in young flax seedlings. By feeding the seedlings with ¹³C₃-p-coumaric acid and isolating isotopically labeled metabolization products prior to the construction of CSPP networks, the biochemical validity of the connections in the network was supported by SIL, independent of spectral similarity or abundance correlation. This method, in combination with multistage mass spectrometry (MSⁿ), allowed confident structural proposals of lignans, neolignans, and hydroxycinnamic acid conjugates, including the presence of newly identified chicoric acid and related tartaric acid esters in flax. High-resolution time-course analyses revealed successive waves of metabolite formation, providing insights into distinct biosynthetic fluxes toward lignans and early lignification intermediates. No evidence was found here for the involvement of chlorogenic or caftaric acid intermediates in chicoric acid biosynthesis in flax, as has been described in other species. Instead, our findings suggest that in flax seedlings, chicoric acid is synthesized through successive hydroxylation steps of p-coumaroyl tartaric acid esters. This work demonstrates the power of combining SIL and CSPP strategies to uncover novel metabolic routes and highlights the nutritional potential of flax sprouts rich in chicoric acid. Full article

In this study, we use coal as a carbon source from Zhangjiamao and doped with different nitrogen sources for co-pyrolysis. Nitrogen-rich tar was successfully prepared, and the content and variety were also increased. From the elemental analysis, the nitrogen content of all the tars was significantly enhanced, among which the nitrogen content of the tars after co-pyrolysis with melamine was enhanced by 5.21%, and the nitrogen content of coke was enhanced by 10.87%. According to the GC/MS results, it was found that the nitrogen compounds in the tar after full pyrolysis were richer and more abundant than those in Py-GC/MS. For the free radical reaction, the reaction process is extremely rapid, and the ¹⁵N substitution product after isotope labeling was successfully captured by adding ¹⁵NH₄Cl for isotope labeling, which can be more intuitively and accurately illustrated from the m/z change. Among them, 26 nitrogen-containing compounds were screened out, which accounted for 66.28% of the content, and they were categorized. It was found that the five-membered nitrogen heterocycles were the most abundant, accounting for 34.88%. In addition, five other nitrogen-containing compounds containing different functional groups and the tar from the co-pyrolysis of tar-rich coal were also analyzed by GC/MS, among which the tar from melamine had the highest content of nitrogen-containing compounds, with 70.48%. Finally, the functional groups of nitrogen-containing compounds were further analyzed by XPS and FTIR, and the results were consistent with those of GC/MS analysis. In this paper, nitrogen-rich tar was prepared by co-pyrolysis of tar-rich coal and nitrogen compounds. This achievement provides a valuable reference for the high-value utilization of coal tar. Full article

Vineyard soils are frequently contaminated with copper due to the use of Cu fungicides to prevent downy mildew. This study investigated the effects of an aerated compost tea (ACT) made from grape pomace and animal manure on the downward transfer of Cu and on the accumulation of Cu in plants in a sandy loam vineyard soil. Crimson clover and pot marigold were grown in a 40 cm soil column with Cu supplied to the surface at loadings representative of those applied in European vineyards, plus additions of ACT. A source of Cu enriched in the stable isotope ⁶⁵Cu was used to distinguish freshly added Cu (fresh Cu) from Cu already present in the soil (aged Cu). ACT increased the concentration of soluble humic substances (SHS) in pore water in the top 7.5 cm of the column, and increased the concentration of Cu, Al, and Fe in pore water in proportion to the concentration of SHS. The transfer of fresh Cu to deeper soil was limited to the top 5 cm, even after the addition of ACT, although fresh Cu reacted slightly more to ACT than aged Cu. ACT had no effect on Cu phytoextraction but increased the concentration of Cu in roots by almost twofold. Relatively more fresh Cu was transferred to plants than aged Cu, primarily due to its preferential accumulation on the surface. The risk associated with the use of ACT on vineyard soils is not that of promoting the downward transfer of Cu, but rather of increasing Cu availability to plants and likely to other living organisms in the topsoil. Full article

Citrinin (CIT) is a nephrotoxic mycotoxin commonly found in a broad range of foods, including cereals, spices, nuts, or Monascus fermentation products. Analyses have shown that CIT is present in processed foods in significantly lower concentrations than in unprocessed materials. Modified forms of CIT arising during food processing may provide an explanation for the discrepancy. This study deals with the thermal stability of CIT and the formation of reaction products of CIT with carbohydrates, followed by toxicological evaluations using cell culture models. HPLC-HRMS degradation curves of CIT heated in different matrix model systems were recorded, and the formation of decarboxycitrinin (DCIT), the main degradation product, was quantified. Additionally, chemical structures of reaction products of CIT with carbohydrates were tentatively identified using MS/MS spectra and stable isotope labelling. Subsequently, the degradation of CIT during biscuit baking was studied, and carbohydrate-bound forms of CIT were detected after enzymatic starch digestion. The formation of DCIT could explain the majority of CIT degradation, but, depending on the process, covalent binding to carbohydrates can also be highly relevant. Cytotoxicity of DCIT in IHKE-cells was found to be lower compared to CIT, while the toxicity as well as the intestinal metabolism of carbohydrate-bound CIT was not evaluated. Full article

China is the world’s largest consumer and second largest producer of bananas. This strong domestic demand consistently provides a reliable income for Chinese banana growers. The geographical origin of food is usually associated with product quality and safety, and this is especially noted for Hainan origin-labeled bananas, which are grown offshore on China’s largest tropical island. Hainan banana is recognized as a premium variety within China’s banana market, but there have been recent impacts on branding, profits, and a reduction in income for banana farmers due to the fraudulent in-market substitution of non-Hainan bananas. In this study, stable isotope and elemental chemometric models were used to differentiate bananas grown in Hainan province (HN) from non-Hainan provinces (NHN). The results showed that HN bananas had a specific isotopic and elemental fingerprint compared to NHN bananas. Bananas sampled from HN and NHN regions showed significant differences in δ¹³C values (HN: −22.2‰ to −27.7‰, NHN: −22.3‰ to −24.3‰), Al content (HN: 0.00 mg/kg to 0.10 mg/kg, NHN: 0.00 mg/kg to 0.02 mg/kg), Na content (HN: 0.00 mg/kg to 0.09 mg/kg, NHN: 0.00 mg/kg to 0.07 mg/kg), and other elements (p < 0.05). Overall, 14 key variables reflecting climate and soil properties were selected from a group of 53 variables to improve a partial least squares discriminant analysis (PLS-DA) chemometric model. The discrimination accuracy of the test set increased from 84.60% to 90.93% after variable reduction. The use of stable isotopes and elements combined with PLS-DA models provided an effective method for distinguishing Chinese HN bananas from NHN bananas and would be useful as a screening or regulatory tool to confirm instances of origin fraud. Full article

Mass spectrometry (MS) is the only instrumental analytical technology that utilizes unique properties of matter, that is, its mass (m) and electrical charge (z). In the magnetic and/or electric fields of mass spectrometers, electrically charged native or chemically modified (millions) endogenous and (thousands) exogenous substances, the analytes, are separated according to their characteristic mass-to-charge ratio (m/z) values. Mass spectrometers coupled to gas chromatographs (GC) or liquid chromatographs (LC), the so-called hyphenated techniques, i.e., GC-MS and LC-MS, respectively, enable reliable determination of the concentration of analytes in complex biological samples such as plasma, serum, and urine. A particular technology is represented by inductively coupled plasma-mass spectrometry (ICP-MS), which is mainly used for the analysis of metal ions. The highest analytical accuracy is reached by using mass spectrometers with high mass resolution (HR) or by tandem mass spectrometers, as it can be realized with quadrupole-type instruments, such as GC-MS/MS and LC-MS/MS, in combination with stable-isotope labeled analytes that serve as internal standards, like a standard weight in scales. GC-MS belongs to the oldest and most advanced instrumental analytical technology. From the very beginning, GC-MS found broad application in basic and applied research sciences. GC-MS has played important roles in discovering biochemical pathways, exploring underlying mechanisms of disease, and establishing new evidence-based pharmacological therapy. In this article, we make an inventory of the use of instrumental mass spectrometry in the life sciences and attempt to provide a perspective study on the future of analytical mass spectrometry in clinical science, mainly focusing on GC-MS and LC-MS. We used information freely available in the scientific database PubMed (retrieved in August–November 2024). Specific search terms such as GC-MS (103,000 articles), LC-MS (113,000 articles), and ICP-MS (14,000 articles) were used in the Title/Abstract in the “PubMed Advanced Search Builder” including filters such as search period (1970–2024). In total, around 103,000 articles on GC-MS, 113,000 articles on LC-MS (113,000), and 14,000 articles on ICP-MS were found. In the period 1995–2023, the yearly publication rate accounted for 3042 for GC-MS articles and 3908 for LC-MS articles (LC-MS/GC-MS ratio, 1.3:1). Our study reveals that GC-MS/MS, LC-MS/MS, and their high-resolution variants are indispensable instrumentations in clinical science including clinical pharmacology, internal and forensic medicine, and doping control. Long-tradition manufacturers of analytical instruments continue to provide increasingly customer-friendly GC-MS and LC-MS apparatus, enabling fulfillment of current requirements and needs in the life sciences. Quantitative GC-MS and GC-MS/MS methods are expected to be used worldwide hand in hand with LC-MS/MS, with ICP-MS closing the gap left for metal ions. The significance of analytical chemistry in clinical science in academia and industry is essential. Full article

L-arginine: glycine amidinotransferase (AGAT) gained academic interest as the rate-limiting enzyme in creatine biosynthesis and its role in the regulation of creatine homeostasis. Of clinical relevance is the diagnosis of patients with AGAT deficiency but also the potential role of AGAT as therapeutic target for the treatment of another creatine deficiency syndrome, guanidinoacetate N-methyltransferase (GAMT) deficiency. Applying a stable isotope-labeled substrate method, we utilized ARG 15N₂ (ARG-δ2) and GLY 13C₂15N (GLY-δ3) to determine the rate of 1,2-13C₂,15N₃ guanidinoacetate (GAA-δ5) formation to assess AGAT activity in various mouse tissue samples and human-derived cells. Following modification and optimization of the assay, we analyzed AGAT activity in several mouse organs. The K_m and V_max of AGAT in mouse kidney for GLY-δ3 were 2.06 mM and 6.48 ± 0.26 pmol/min/mg kidney, and those for ARG-δ2, they were 2.67 mM and 2.17 ± 0.49 pmol/min/mg kidney, respectively. Our results showed that mouse kidneys had the highest levels of enzymatic activity, followed by brain and liver, with 4.6, 1.8, and 0.4 pmol/min/mg tissue, respectively. Both the heart and muscle had no detectable levels of AGAT activity. We noted that due to interference with arginase in the liver, performing the enzyme assay in liver homogenates required the addition of Nor-NOHA, an arginase inhibitor. In immortalized human cell lines, we found the highest levels of AGAT activity in RH30 cells, followed by HepaRG, HAP1, and HeLa cells. AGAT activity was readily detectable in lymphoblasts and leukocytes from healthy controls. In our assay, AGAT activity was not detectable in HEK293 cells, in human fibroblasts, and in the lymphoblasts of a patient with AGAT deficiency. Our results demonstrate that this enzyme assay is capable of accurately quantifying AGAT activity from both tissues and cells for diagnostic purposes and research. Full article

Background: Acute and chronic pre-traumatic cigarette smoke exposure increases morbidity and mortality after trauma and hemorrhage. In mice with a genetic deletion of the H₂S-producing enzyme cystathione-γ-lyase (CSE^−/−), providing exogenous H₂S using sodium thiosulfate (Na₂S₂O₃) improved organ function after chest trauma and hemorrhagic shock. Therefore, we evaluated the effect of Na₂S₂O₃ during resuscitation from blunt chest trauma and hemorrhagic shock on CSE^−/− mice with pre-traumatic cigarette smoke (CS) exposure. Since H₂S is well established as being able to modify energy metabolism, a specific focus was placed on whole-body metabolic pathways and mitochondrial respiratory activity. Methods: Following CS exposure, the CSE^−/− mice underwent anesthesia, surgical instrumentation, blunt chest trauma, hemorrhagic shock for over 1 h (target mean arterial pressure (MAP) ≈ 35 ± 5 mmHg), and resuscitation for up to 8 h comprising lung-protective mechanical ventilation, the re-transfusion of shed blood, fluid resuscitation, and continuous i.v. noradrenaline (NoA) to maintain an MAP ≥ 55 mmHg. At the start of the resuscitation, the mice randomly received either i.v. Na₂S₂O₃ (0.45 mg/g_bodyweight; n = 14) or the vehicle (NaCl 0.9%; n = 11). In addition to the hemodynamics, lung mechanics, gas exchange, acid–base status, and organ function, we quantified the parameters of carbohydrate, lipid, and protein metabolism using a primed continuous infusion of stable, non-radioactive, isotope-labeled substrates (gas chromatography/mass spectrometry) and the post-mortem tissue mitochondrial respiratory activity (“high-resolution respirometry”). Results: While the hemodynamics and NoA infusion rates did not differ, Na₂S₂O₃ was associated with a trend towards lower static lung compliance (p = 0.071) and arterial PO₂ (p = 0.089) at the end of the experiment. The direct, aerobic glucose oxidation rate was higher (p = 0.041) in the Na₂S₂O₃-treated mice, which resulted in lower glycemia levels (p = 0.050) and a higher whole-body CO₂ production rate (p = 0.065). The mitochondrial respiration in the heart, kidney, and liver tissue did not differ. While the kidney function was comparable, the Na₂S₂O₃-treated mice showed a trend towards a shorter survival time (p = 0.068). Conclusions: During resuscitation from blunt chest trauma and hemorrhagic shock in CSE^−/− mice with pre-traumatic CS exposure, Na₂S₂O₃ was associated with increased direct, aerobic glucose oxidation, suggesting a switch in energy metabolism towards preferential carbohydrate utilization. Nevertheless, treatment with Na₂S₂O₃ coincided with a trend towards worsened lung mechanics and gas exchange, and, ultimately, shorter survival. Full article

Globalization and free market dynamics have significantly impacted state economies, particularly in the wine industry. These forces have introduced greater diversity in wine products but have also heightened the risk of food fraud, especially in high-value commodities like wine. Due to its market value and the premium placed on quality, wine is frequently subject to adulteration. This issue is often addressed through regulatory trademarks on wine labels, such as Protected Designation of Origin (PDO) and Protected Geographic Indication (PGI). In this context, the metabolic profiles (organic acids, carbohydrates, and phenols) and stable isotope signatures (δ¹³C, δ¹⁸O, D/H_I, and D/H_II) of red and white wines from four agroclimatically similar regions were examined. The study explored how factors such as grape variety, harvest year, and geographical origin affect wine composition, with a particular focus on distinguishing samples from cross-border areas. Multivariate statistical analysis was used to assess the variability in wine composition and to identify distinct groups of samples. Preliminary results revealed that organic acids and volatile compounds were found in lower concentrations than carbohydrates but were significantly higher than phenols, with levels ranging between 1617 mg/L and 6258 mg/L. Carbohydrate content in the wines varied from 8285 mg/L to 14662 mg/L. Principal Component Analysis (PCA) indicated certain separation trends based on the variance in carbohydrates (e.g., fructose, glucose, galactose) and isotopic composition. However, Discriminant Analysis (DA) provided clear distinctions based on harvest year, variety, and geographical origin. Full article

Background: Our study presented a novel LC-MS/MS method for the simultaneous quantification of α-tocopherol (α-TOH) and its phase II metabolites, α-13′-COOH and α-13′-OH, in human serum using deuterium-labeled internal standards (d₆-α-TOH, d₆-α-13′-COOH, d₆-α-13′-OH). Methods: The method addresses the analytical challenge posed by the significantly different concentration ranges of α-TOH (µmol/L) and its metabolites (nmol/L). Previous methods quantified these analytes separately, which caused an increase in workflow complexity. Results: Key features include the synthesis of stable isotope-labeled standards and the use of a pentafluorophenyl-based core-shell chromatography column for baseline separation of both α-TOH and its metabolites. Additionally, solid phase extraction (SPE) with a HybridSPE^® material provides a streamlined sample preparation, enhancing analyte recovery and improving sensitivity. By utilizing deuterium-labeled standards, the method compensates for matrix effects and ion suppression. This new approach achieves precise and accurate measurements with limits of detection (LOD) and quantification (LOQ), similar to previous studies. Calibration, accuracy, and precision parameters align well with the existing literature. Conclusions: Our method offers significant advantages in the simultaneous analysis of tocopherol and its metabolites despite concentration differences spanning up to three orders of magnitude. In contrast to earlier studies, which required separate sample preparations and analytical techniques for tocopherol and its metabolites, our approach streamlines this process. The use of a solid-phase extraction procedure allows for parallel sample preparation. This not only enhances efficiency but also significantly accelerates pre-analytical workflows, making the method highly suitable for large-scale studies. Full article

Acute lung Injury leads to alterations in surfactant lipid composition and metabolism. Although several mechanisms contribute to dysregulated surfactant metabolism, studies investigating in vivo surfactant metabolism are limited. The aim of this study is to characterise surfactant phospholipid composition and flux utilising a stable isotope labelling technique in mechanically ventilated paediatric patients. Paediatric patients (<16 years of age) received 3.6 mg/kg intravenous methyl-D₉-choline chloride followed by the endotracheal instillation of 100 mg/kg of exogenous surfactant after 24 h. Bronchioalveolar fluid samples were taken at baseline and 12, 24, 36, 48, 72 and 96 h after methyl-D₉-choline infusion. Nine participants (median age of 48 days) were recruited. The primary phosphatidylcholine (PC) composition consisted of PC16:0/16:0 or DPPC (32.0 ± 4.5%). Surfactant supplementation resulted in a 30% increase in DPPC. Methyl-D₉ PC enrichment was detected after 12 h and differed significantly between patients, suggesting variability in surfactant synthesis/secretion by the CDP-choline pathway. Peak enrichment was achieved (0.94 ± 0.15% of total PC) at 24 h after methyl-D₉-choline infusion. There was a trend towards reduced enrichment with the duration of mechanical ventilation prior to study recruitment; however, this was not statistically significant (p = 0.19). In this study, we demonstrated the fractional molecular composition and turnover of surfactant phospholipids, which was highly variable between patients. Full article

Passive smoking from environmental tobacco smoke not only increases the risk of lung cancer and cardiovascular disease but may also be a stressor triggering neuropsychiatric and other disorders. To prevent these diseases, understanding the relationship between passive smoking and stress is vital. In this study, we developed a simple and sensitive method to simultaneously measure nicotine (Nic) and cotinine (Cot) as tobacco smoke exposure biomarkers, and cortisol (CRT), serotonin (5-HT), melatonin (MEL), dopamine (DA), and oxytocin (OXT) as stress-related biomarkers. These were extracted and concentrated from saliva by in-tube solid-phase microextraction (IT-SPME) using a Supel-Q PLOT capillary as the extraction device, then separated and detected within 6 min by liquid chromatography–tandem mass spectrometry (LC−MS/MS) using a Kinetex Biphenyl column (Phenomenex Inc., Torrance, CA, USA). Limits of detection (S/N = 3) for Nic, Cot, CRT, 5-HT, MEL, DA, and OXT were 0.22, 0.12, 0.78, 0.39, 0.45, 1.4, and 3.7 pg mL⁻¹, respectively, with linearity of calibration curves in the range of 0.01–25 ng mL⁻¹ using stable isotope-labeled internal standards. Intra- and inter-day reproducibilities were under 7.9% and 14.6% (n = 5) relative standard deviations, and compound recoveries in spiked saliva samples ranged from 82.1 to 106.6%. In thirty nonsmokers, Nic contents positively correlated with CRT contents (R² = 0.5264, n = 30), while no significant correlation was found with other biomarkers. The standard deviation of intervals between normal beats as the standard measure of heart rate variability analysis negatively correlated with CRT contents (R² = 0.5041, n = 30). After passive smoke exposure, Nic levels transiently increased, Cot and CRT levels rose over time, and 5-HT, DA, and OXT levels decreased. These results indicate tobacco smoke exposure acts as a stressor in nonsmokers. Full article

The shiitake mushroom has gained popularity in the last decade, ranking second in the world for mushrooms consumed, providing consumers with a wide variety of nutritional and healthy benefits. It is often not clear the origin of these mushrooms, so it becomes of great importance to the consumers. In this research, different machine learning algorithms were developed to determine the geographical origin of shiitake mushrooms (Lentinula edodes) consumed in Korea, based on experimental data reported in the literature (δ¹³C, δ¹⁵N, δ¹⁸O, δ³⁴S, and origin). Regarding the origin of shiitake in three categories (Korean, Chinese, and mushrooms from Chinese inoculated sawdust blocks), the random forest model presents the highest accuracy value (0.940) and the highest kappa value (0.908) for the validation phase. To determine the origin of shiitake mushrooms in two categories (Korean and Chinese, including mushrooms from Chinese inoculated sawdust blocks in the latter ones), the support vector machine model is chosen as the best model due to the high accuracy (0.988) and kappa (0.975) values for the validation phase. Finally, to determine the origin in two categories (Korean and Chinese, but this time including the mushrooms from Chinese inoculated sawdust blocks in the Korean ones), the best model is the random forest due to its higher accuracy value (0.952) in the validation phase (kappa value of 0.869). The accuracy values in the testing phase for the best selected models are acceptable (between 0.839 and 0.964); therefore, the predictive capacity of the models could be acceptable for their use in real applications. This allows us to affirm that machine learning algorithms would be suitable modeling instruments to determine the geographical origin of shiitake. Full article

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach—quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques. Full article

Aquaculture contributes to the sustainable development of food security, marine resource conservation, and economy. Shifting aquaculture feed from fish meal and oil to terrestrial plant derivatives may result in cost savings. However, many carnivorous fish cannot be sustained on plant-derived materials, necessitating the need for the identification of important factors for farmed fish growth and the identification of whether components derived from terrestrial plants can be used in feed. Herein, we focused on the carnivorous fish leopard coral grouper (P. leopardus) to identify the essential growth factors and clarify their intake timing from feeds. Furthermore, we evaluated the functionality of starch, which are easily produced by terrestrial plants. Results reveal that carbohydrates, which are not considered essential for carnivorous fish, can be introduced as a major part of an artificial diet. The development of artificial feed using starch offers the possibility of increasing the growth of carnivorous fish in aquaculture. Full article