Metabolomics on Apple (Malus domestica) Cuticle-Search for Authenticity Markers.

The profile of secondary metabolites present in the apple cuticular layer is not only characteristic of a particular apple cultivar; it also dynamically reflects various external factors in the growing environment. In this study, the possibility of authenticating apple samples by analyzing their cuticular layer extracts was investigated. Ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) was employed for obtaining metabolomic fingerprints. A total of 274 authentic apple samples from four cultivars harvested in the Czech Republic and Poland between 2020 and 2022 were analyzed. The complex data generated, processed using univariate and multivariate statistical methods, enabled the building of classification models to distinguish apple cultivars as well as their geographical origin. The models showed very good performance in discriminating Czech and Polish samples for three out of four cultivars: "Gala", "Golden Delicious" and "Idared". Moreover, the validity of the models was tested over several harvest seasons. In addition to metabolites of the triterpene biosynthetic pathway, the diagnostic markers were mainly wax esters. "Jonagold", which is known to be susceptible to mutations, was the only cultivar for which an unambiguous classification of geographical origin was not possible.

Plasma lipidome differences in patients with and without significant carotid plaque.

BACKGROUND: Atherosclerosis is a major cause of ischemic stroke, and early detection of advanced atherosclerosis in the carotid artery is important for reducing morbidity and mortality. What is even more important is not only detection of atherosclerosis but early determination whether the patients are at high risk of an event with adverse effects as the size of the plaque does not necessarily reflect its potential to trigger such events. AIM: We studied whether plasma lipidomics profile can be used as a diagnostic tool for stratification of stable or unstable plaques without the need of removing the carotid plaque. METHODS: This study used liquid chromatography high-resolution tandem mass spectrometry lipidomics to characterize lipid profiles in patients' plasma and found that patients with significant and complicated (vulnerable) atherosclerotic plaque had distinct lipid profiles compared to those with insignificant plaques. RESULTS: The lipid classes that were most predictive of vulnerable plaque were lysophosphoethanolamines, fatty acyl esters of hydroxy fatty acids, free fatty acids, plasmalogens, and triacylglycerols. Most of these compounds were found decreased in plasma of patients with unstable plaques which enabled sufficient performance of a statistical model used for patient stratification. CONCLUSIONS: Plasma lipidomes measured by liquid chromatography-mass spectrometry show differences in patients with stable and unstable carotid plaques, therefore these compounds could potentially be used as biomarkers for unstable plaque in future clinical diagnosis.

The Ameliorating Effects of n-3 Polyunsaturated Fatty Acids on Liver Steatosis Induced by a High-Fat Methionine Choline-Deficient Diet in Mice.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is associated with abnormalities of liver lipid metabolism. On the contrary, a diet enriched with n-3 polyunsaturated fatty acids (n-3-PUFAs) has been reported to ameliorate the progression of NAFLD. The aim of our study was to investigate the impact of dietary n-3-PUFA enrichment on the development of NAFLD and liver lipidome. Mice were fed for 6 weeks either a high-fat methionine choline-deficient diet (MCD) or standard chow with or without n-3-PUFAs. Liver histology, serum biochemistry, detailed plasma and liver lipidomic analyses, and genome-wide transcriptome analysis were performed. Mice fed an MCD developed histopathological changes characteristic of NAFLD, and these changes were ameliorated with n-3-PUFAs. Simultaneously, n-3-PUFAs decreased serum triacylglycerol and cholesterol concentrations as well as ALT and AST activities. N-3-PUFAs decreased serum concentrations of saturated and monounsaturated free fatty acids (FAs), while increasing serum concentrations of long-chain PUFAs. Furthermore, in the liver, the MCD significantly increased the hepatic triacylglycerol content, while the administration of n-3-PUFAs eliminated this effect. Administration of n-3-PUFAs led to significant beneficial differences in gene expression within biosynthetic pathways of cholesterol, FAs, and pro-inflammatory cytokines (IL-1 and TNF-α). To conclude, n-3-PUFA supplementation appears to represent a promising nutraceutical approach for the restoration of abnormalities in liver lipid metabolism and the prevention and treatment of NAFLD.

European food safety research: An explorative study with funding experts' consultation.

The European research area exhibits considerable opacity and fragmentation in food safety research funding and organizational structures, impeding the exploitation of existing research potential across European countries. Given that food safety is inherently linked to the societal challenges of our time, identifying and removing existing barriers to research funding in this area is crucial. Towards investigating this matter, interviews were conducted with funding bodies from six European countries to assess key issues related to research funding in general and food safety in particular. Funding experts were then invited to a workshop to jointly discuss the challenges identified and explore strategies to address them. Evaluation of the food safety research funding situation in selected European countries revealed both convergences and significant differences among national funding bodies. Engaging with funding experts provided invaluable insights into the issues encountered with research funding, such as inadequate call management staff or insufficient research funds, culminating in a set of recommendations for action to remedy the situation.

A lipidomic and metabolomic signature of a very low-carbohydrate high-fat diet and high-intensity interval training: an additional analysis of a randomized controlled clinical trial.

INTRODUCTION: Regular physical activity and dietary variety are modifiable and influential factors of health outcomes. However, the cumulative effects of these behaviors are not well understood. Metabolomics may have a promising research potential to extend our knowledge and use it in the attempts to find a long-term and sustainable personalized approach in exercise and diet recommendations. OBJECTIVE: The main aim was to investigate the effect of the 12 week very low carbohydrate high fat (VLCHF) diet and high-intensity interval training (HIIT) on lipidomic and metabolomic profiles in individuals with overweight and obesity. METHODS: The participants (N = 91) were randomly allocated to HIIT (N = 22), VLCHF (N = 25), VLCHF + HIIT (N = 25) or control (N = 19) groups for 12 weeks. Fasting plasma samples were collected before the intervention and after 4, 8 and 12 weeks. The samples were then subjected to untargeted lipidomic and metabolomic analyses using reversed phase ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry. RESULTS: The VLCHF diet affected plasma lipids considerably while the effect of HIIT was unremarkable. Already after 4 weeks of intervention substantial changes of plasma lipids were found in both VLCHF diet groups. The changes persisted throughout the entire 12 weeks of the VLCHF diet. Specifically, acyl carnitines, plasmalogens, fatty acyl esters of hydroxy fatty acid, sphingomyelin, ceramides, cholesterol esters, fatty acids and 4-hydroxybutyric were identified as lipid families that increased in the VLCHF diet groups whereas lipid families of triglycerides and glycerophospholipids decreased. Additionally, metabolomic analysis showed a decrease of theobromine. CONCLUSIONS: This study deciphers the specific responses to a VLCHF diet, HIIT and their combination by analysing untargeted lipidomic and metabolomic profile. VLCHF diet caused divergent changes of plasma lipids and other metabolites when compared to the exercise and control group which may contribute to a better understanding of metabolic changes and the appraisal of VLCHF diet benefits and harms. CLINICAL TRIAL REGISTRY NUMBER: NCT03934476, registered 1st May 2019 https://clinicaltrials.gov/ct2/show/NCT03934476?term=NCT03934476&draw=2&rank=1 .

Pesticide residues in fresh and processed edible mushrooms from Czech markets.

The occurrence of pesticide residues, their metabolites and degradation products in 49 edible mushrooms (fresh, dried, canned and frozen) was investigated. Using QuEChERS extraction, followed by liquid and gas chromatography coupled with tandem mass spectrometry, the samples collected from Czech markets were tested for 427 analytes. A total of 21 pesticide residues, their metabolites and pesticide synergists were found in quantifiable concentrations. The most frequently detected pesticide residues included prochloraz and its metabolites, metrafenone and carbendazim. Two mushroom samples were found to be in violation of the maximum residue level. In addition, the fate of 13 pesticides during the processing of fresh mushrooms was investigated. The processing factors calculated from the data ranged from 6.0 to 12.3 for drying and from 0.3 to 1.3 for deep-freezing. The results showed that most of the pesticide residues tested were stable under the conditions of mushroom processing.

Metabolomic and transcriptomic analyses identify external conditions and key genes underlying high levels of toxic glycoalkaloids in tubers of stress-sensitive potato cultivars.

Introduction: High levels of toxic steroidal glycoalkaloids (SGAs) in potato tubers constitute a recognized food quality problem. Tuber SGA levels vary between potato cultivars and can increase after post-harvest stresses such as wounding and light exposure. A few cultivars, e.g., 'Magnum Bonum' and 'Lenape,' have been withdrawn from commercial sales due to excessive SGA levels during some cultivation years. However, these sudden SGA increases are diffucult to predict, and their causes are not understood. To identify external and genetic factors that underlie sudden SGA increases in certain potato cultivars, we have here in a 2-year study investigated 'Magnum Bonum' and five additional table potato cultivars for their SGA levels after wounding and light exposure. Results and methods: Results showed that 'Magnum Bonum' has an unusual strong SGA response to light exposure, but not to wounding, whereas 'Bintje' displayed an opposite regulation. Levels of calystegine alkaloids were not significantly altered by treatments, implicating independent metabolic regulation of SGA and calystegine levels also under conditions of high SGA accumulation. Metabolomic and transcriptomic analyses identified a small number of key genes whose expression correlated with SGA differences between cultivars. Overexpression of two key genes in transgenic low-SGA potato cultivars increased their leaf SGA levels significantly. Discussion: The results show that a strong response to light can underlie the SGA peaks that occasionally occur in certain potato cultivars and indicate that a between-cultivar variation in the expression of single SGA key genes can account for cultivar SGA differerences. We propose that current attempts to mitigate the SGA hazard will benefit from an increased consideration of cultivar-dependent SGA responses to post-harvest conditions, particularly light exposure. The identified key SGA genes can now be used as a molecular tool in this work.

Enhanced production of select phytocannabinoids in medical Cannabis cultivars using microbial consortia.

The root microbiome of medical cannabis plants has been largely unexplored due to past legal restrictions in many countries. Microbes that live on and within the tissue of Cannabis sativa L. similar to other plants, provide advantages such as stimulating plant growth, helping it absorb minerals, providing protection against pathogen attacks, and influencing the production of secondary metabolites. To gain insight into the microbial communities of C. sativa cultivars with different tetrahydrocannabinol (THC) and cannabidiol (CBD) profiles, a greenhouse trial was carried out with and without inoculants added to the growth substrate. Illumina MiSeq metabarcoding was used to analyze the root and rhizosphere microbiomes of the five cultivars. Plant biomass production showed higher levels in three of five cultivars inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis and microbial suspension. The blossom dry weight of the cultivar THE was greater when inoculated with R. irregularis and microbial suspension than with no inoculation. Increasing plant biomass and blossom dry weight are two important parameters for producing cannabis for medical applications. In mature Cannabis, 12 phytocannabinoid compounds varied among cultivars and were affected by inoculants. Significant differences (p ≤ 0.01) in concentrations of cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), and cannabigerolic acid (CBGA) were observed in all Cannabis cultivars when amended with F, K1, and K2 inoculants. We found microbes that were shared among cultivars. For example, Terrimicrobium sp., Actinoplanes sp., and Trichoderma reesei were shared by the cultivars ECC-EUS-THE, CCL-ECC, and EUS-THE, respectively. Actinoplanes sp. is a known species that produces phosphatase enzymes, while Trichoderma reesei is a fungal train that produces cellulase and contributes to organic matter mineralization. However, the role of Terrimicrobium sp. as an anaerobic bacterium remains unknown. This study demonstrated that the use of inoculants had an impact on the production of phytocannabinoids in five Cannabis cultivars. These inoculants could have useful applications for optimizing cannabis cultivation practices and increasing the production of phytocannabinoids.

Detection of botanical adulterants in saffron powder.

Saffron is a unique spice obtained by drying stigmas of saffron flowers (Crocus sativus L.). Due to its high price, economically motivated adulteration occurs relatively often. The presented study aimed to develop an effective strategy for the detection of the following potential botanical adulterants used for a saffron substitution or dilution: safflower (Carthamus tinctorius L.), calendula (Calendula officinalis L.), turmeric (Curcuma longa L.), achiote (Bixa orellana L.), red pepper (Capsicum spp.), mountain arnica (Arnica montana L.), beet (Beta vulgaris L.), and pomegranate (Punica granatum L.). A non-target screening strategy based on ultra-high performance reverse-phase liquid chromatography coupled to tandem high-resolution mass spectrometry (UHPLC-HRMS/MS) was employed for the analysis of an aqueous ethanol plant extract. By using multivariate statistical methods, principal components analysis (PCA), and partial least squares discriminant analysis (PLS-DA), for processing the generated "chemical fingerprints," metabolites unique to the investigated plants could be identified. To enable routine saffron authenticity control by target screening, an internal spectral database was developed; currently, it involves 82 unique markers. In this way, the detection addition as low as 1% (w/w) of all analyzed botanical adulterants in admixture with saffron was possible. The developed method was used to control 7 saffron powder samples from the Czech market, and none of the monitored adulterants were confirmed.

Effective isolation of cannabidiol and cannabidiolic acid free of psychotropic phytocannabinoids from hemp extract by fast centrifugal partition chromatography.

Cannabidiol (CBD), together with its precursor cannabidiolic acid (CBDA), is the major phytocannabinoid occurring in most hemp cultivars. To ensure the safe use of these compounds, their effective isolation from hemp extract is required, with special emphasis on the elimination of ∆9-tetrahydrocannabinol (∆9-THC) and ∆9-tetrahydrocannabinolic acid (∆9-THCA-A). In this study, we demonstrate the applicability of fast centrifugal partition chromatography (FCPC) as a challenging format of counter-current preparative chromatography for the isolation of CBD and CBDA free of psychotropic compounds that may occur in Cannabis sativa L. plant extracts. Thirty-eight solvent mixtures were tested to identify a suitable two-phase system for this purpose. Based on the measured partition coefficients (KD) and separation factors (α), the two-phase system consisting of n-heptane:ethyl acetate:ethanol:water (1.5:0.5:1.5:0.5; v:v:v:v) was selected as an optimal solvent mixture. Employing UHPLC-HRMS/MS for target analysis of collected fractions, the elution profiles of 17 most common phytocannabinoids were determined. Under experimental conditions, the purity of isolated CBD and CBDA was 98.9 and 95.1% (w/w), respectively. Neither of ∆9-THC nor of ∆9-THCA-A were present; only trace amounts of other biologically active compounds contained in hemp extract were detected by screening against in-house spectral library using UHPLC-HRMS.

POPs and PAHs in fish oil-based food supplements at the Czech market.

The objectives of this study were to assess concentrations of three groups of persistent organic pollutants (POPs) and polycyclic aromatic hydrocarbons (PAHs) in 44 fish oil-based food supplements, to estimate their daily intake by consumers and, to evaluate the compliance of the oil samples with the oil origin declarations (cod liver oil or fish oil). The concentrations of ∑PCBs (7 congeners), OCPs (19 compounds, represented mainly by ∑DDTs), ∑PBDEs (10 congeners), and ∑PAHs (16 compounds) found in samples ranged between 0.15-55.7 µg kg-1, 0.93-72.8 µg kg-1, 0.28-27.5 µg kg-1, and 0.32-51.9 µg kg-1, respectively. Besides, the authenticity of the oils was assessed based on the fingerprints obtained by DART-HRMS, an ambient mass spectrometry technique. Four samples declared as fish oil were probably prepared from cod liver oil, which is much cheaper. Furthermore, these samples contained elevated concentrations of halogenated POPs when compared to supplements produced from fish oil.

Modulation of the bacterial virulence and resistance by well-known European medicinal herbs.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L., Sambucus nigra L., Matricaria chamomilla L., Agrimonia eupatoria L., Fragaria vesca L. and Malva sylvestris L. are plants that have a long tradition in European folk medicine. To this day, they are part of medicinal teas or creams that help with the healing of skin wounds and the treatment of respiratory or intestinal infections. However, so far these plants have not been investigated more deeply than in their direct antibacterial effect. AIM OF THE STUDY: Our research is focused on adjuvants that inhibit the mechanism of antibiotic resistance or modulate bacterial virulence. Based on a preliminary screening of 52 European herbs, which commonly appear as part of tea blends or poultice. Six of them were selected for their ability to revert the resistant phenotype of nosocomial bacterial strains. METHODS: Herbs selected for this study were obtained from commercially available sources. For the extraction of active compounds ethanol was used. Modulation of virulence was observed as an ability to inhibit bacterial cell-to-cell communication using two mutant sensor strains of Vibrio campbellii. Biofilm formation, and planktonic cell adhesion was measured using a static antibiofilm test. Ethidium bromide assay was used to checked the potential of inhibition bacterial efflux pumps. The antibacterial activities of the herbs were evaluated against resistant bacterial strains using macro dilution methods. RESULTS: Alcohol extracts had antibacterial properties mainly against Gram-positive bacteria. Of all of them, the highest antimicrobial activity demonstrated Malva sylvestris, killing both antibiotic resistant bacteria; Staphylococcus aureus with MIC of 0.8 g/L and Pseudomonas aeruginosa 0.7 g/L, respectively. Fragaria vesca extract (0.08 g/L) demonstrated strong synergism with colistin (4 mg/L) in modulating the resistant phenotype to colistin of Pseudomonas aeruginosa. Similarly, the extract of S. officinalis (0.21 g/L) reverted resistance to gentamicin (1 mg/L) in S. aureus. However, Sambucus nigra and Matricaria chamomilla seem to be a very promising source of bacterial efflux pump inhibitors. CONCLUSION: The extract of F. vesca was the most active. It was able to reduce biofilm formation probably due to the ability to decrease bacterial quorum sensing. On the other hand, the activity of S. nigra or M. chamomilla in reducing bacterial virulence may be explained by the ability to inhibit bacterial efflux systems. All these plants have potential as an adjuvant for the antibiotic treatment.

External Quality Assurance Schemes (EQUASs) and Inter-laboratory Comparison Investigations (ICIs) for human biomonitoring of polycyclic aromatic hydrocarbon (PAH) biomarkers in urine as part of the quality assurance programme under HBM4EU.

Polycyclic aromatic hydrocarbons (PAHs) were included as priority substances for human biomonitoring (HBM) in the European Human Biomonitoring Initiative (HBM4EU), which intended to harmonise and advance HBM across Europe. For this project, a specific Quality Assurance and Quality Control (QA/QC) programme applying Inter-laboratory Comparison Investigations (ICIs) and External Quality Assurance Schemes (EQUASs) was developed to ensure the comparability and accuracy of participating analytical laboratories. This paper presents the results of four ICI/EQUAS rounds for the determination of 13 PAH metabolites in urine, i.e. 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene, 2-, 3- and 9-hydroxyfluorene, 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene. However, 4 PAH metabolites could not be evaluated as the analytical capacity of participating laboratories was too low. Across all rounds and biomarkers, 86% of the participants achieved satisfactory results, although low limits of quantification were required to quantify the urinary metabolites at exposure levels of the general population. Using high-performance liquid or gas chromatography coupled with mass spectrometry (HPLC-MS; GC-MS) and isotope dilution for calibration as well as performing an enzymatic deconjugation step proved to be favourable for the accurate determination of PAHs in urine. Finally, the HBM4EU QA/QC programme identified an international network of laboratories providing comparable results in the analysis of urinary PAH biomarkers, although covering all parameters initially selected was still too challenging.

Differentiation of sea buckthorn syrups processed by high pressure, pulsed electric fields, ohmic heating, and thermal pasteurization based on quality evaluation and chemical fingerprinting.

Introduction: Impact of processing on product characteristics, sustainability, traceability, authenticity, and public health along the food chain becomes more and more important not only to the producer but also to the customer and the trust of a consumer toward a brand. In recent years, the number of juices and smoothies containing so called super foods or fruits, which have been "gently pasteurized," has increased significantly. However, the term "gentle pasteurization" related to the application of emerging preservation technologies such as pulsed electric fields (PEF), high pressure processing (HPP) or ohmic heating (OH) is not clearly defined. Methods: Therefore, the presented study investigated the influence of PEF, HPP, OH, and thermal treatment on quality characteristics and microbial safety of sea buckthorn syrup. Syrups from two different varieties were investigated under the following conditions HPP (600 MPa 4-8 min), OH (83°C and 90°C), PEF (29.5 kV/cm, 6 µs, 100 Hz), and thermal (88°C, hot filling). Analyses to test the influence on quality parameters like ascorbic acid (AA), flavonoids, carotenoids, tocopherols, antioxidant activity; metabolomical/chemical profiling (fingerprinting) via U-HPLC-HRMS/MS (here especially flavonoids and fatty acids); sensory evaluation, as well as microbial stability including storage, were conducted. Results and discussion: Independent from the treatment, the samples were stable over 8 weeks of storage at 4°C. The influence on the nutrient content [Ascorbic acid (AA), total antioxidant activity (TAA), total phenolic compounds (TPC), tocopherols (Vit E)] was similar for all tested technologies. Employing statistical evaluation Principal Component Analysis (PCA) a clear clustering based on the processing technologies was observed. Flavonoids as well as fatty acids were significantly impacted by the type of used preservation technology. This was obvious during the storage time of PEF and HPP syrups, where enzyme activity was still active. The color as well as taste of the syrups were found to be more fresh-like for the HPP treated samples.

Tremorgenic mycotoxin poisoning in a dog: A case report.

An eleven-year-old Pit Bull Terrier was presented to the veterinary practice with an acute onset of whole-body seizures. The clinical signs developed in a garden where the dog was kept that morning. There was a suspicion of tremorgenic mycotoxin poisoning by compost as the dog had vomited parts of compost right before the onset of the seizures and there was a pile of compost located in the garden. The dog underwent immediate decontamination following supportive treatment and recovered fully within 24 h of intensive care. The samples of the vomit and parts of the compost were cultivated. In the sample of the vomit, Penicillium sp. was found. Subsequently, tremorgenic mycotoxins paxilline, penitrem A and roquefortine C were determined chromatographically at significant concentrations in the vomit and a growth medium with cultivated Penicillium sp. The aim of this work is to describe the complex therapeutic and diagnostic approach to the patient with a suspected tremorgenic mycotoxin poisoning where a combination of mycological and chromatographic analyses was used to confirm the diagnosis. To the best of our knowledge, this is the first confirmed case of canine tremorgenic mycotoxicosis in the Czech Republic and the first reported case of paxilline poisoning in a dog.

The In Vitro Inhibitory Effect of Selected Asteraceae Plants on Pancreatic Lipase Followed by Phenolic Content Identification through Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS).

Pancreatic lipase (PNLIP, EC 3.1.1.3) plays a pivotal role in the digestion of dietary lipids, a metabolic pathway directly related to obesity. One of the effective strategies in obesity treatment is the inhibition of PNLIP, which is possible to be achieved by specific phenolic compounds occurring in high abundance in some plants. In this study, a multidisciplinary approach is presented investigating the PNLIP inhibitory effect of 33 plants belonging in the Asteraceae botanical family. In the first stage of the study, a rapid and cost-efficient PNLIP assay in a 96-microwell plate format was developed and important parameters were optimized, e.g., the enzyme substrate. Upon PNLIP assay optimization, aqueous and dichloromethane Asteraceae plant extracts were tested and a cut-off inhibition level was set to further analyze only the samples with a significant inhibitory effect (inhibitory rate > 40%), using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) method. Specifically, a metabolomic suspect screening was performed and 69 phenolic compounds were tentatively identified, including phenolic acids, flavonoids, flavonoid-3-O-glycosides, and flavonoid-7-O-glycosides, amongst others. In the case of aqueous extracts, phytochemicals known for inducing PNLIP inhibitory effect, e.g., compounds containing galloyl molecules or caffeoylquinic acids, were monitored in Chrysanthemum morifolium, Grindella camporum and Hieracium pilosella extracts. All in all, the presented approach combines in vitro bioactivity measurements to high-end metabolomics to identify phenolic compounds with potential medicinal and/or dietary applications.

Formation of Previously Undescribed Δ7-Phytosterol Oxidation Products and Tocopherylquinone Adducts in Pumpkin Seed Oil during Roasting, Screw-Pressing, and Simulated Culinary Processing at Elevated Temperatures.

The influence of pumpkin seed roasting conditions (110-140 °C) and screw-pressing on the formation of previously undescribed Δ7-phytosterol oxidation products and tocopherylquinone adducts with nucleophilic phosphatidylethanolamine species was investigated. The roasting process of pumpkin seed paste at a temperature above 120 °C for 30 min considerably enhanced the formation of Δ7-oxysterols. Targeted analysis [electron impact mass spectrometry (MS), 1D-nuclear magnetic resonance] led to the identification of five novel markers of pumpkin paste roasting, among which (3ß,5α,22E,24S)-stigmasta-7,22-dien-6-one-3-ol (6-oxo-α-spinasterol), stereoisomers of (3ß,5α,22E)-7,8-epoxystigmast-22-en-3-ol (7,8-epoxy-α-spinasterol), and (3ß,5α)-22,23-epoxystigmast-7-en-3-ol (7,8-epoxy-α-spinasterol) were reported in edible oils for the first time. Simulated culinary processing provided novel stereoisomers of (3ß,5α,22E)-stigmasta-7,22-dien-3,6-diol, unusual (3ß,5α,22E)-stigmasta-7,22-dien-6,15-dione-3-ol, and (5α,22E)-stigmasta-7,22-dien-3-one accompanied by minor stereoisomers of (3ß,5α)-7,8;22,23-diepoxystigmastan-3-ol. Moreover, a clear relationship between the pumpkin seed oil stability index and synergistic effect of glycerophospholipids with present tocochromanols was found. High-resolution atmospheric pressure chemical ionization-MS experiments clearly demonstrated the formation of various γ-tocopherylquinone adducts with primary amines, namely, octylamine. The mitigation strategy of potentially detrimental oxysterols from pumpkin seed oil included optimization of processing parameters while maintaining the formation of desirable sensory-active compounds.

Isoprenaline modified the lipidomic profile and reduced ß-oxidation in HL-1 cardiomyocytes: In vitro model of takotsubo syndrome.

Recent studies have suggested a pathogenetic link between impaired mitochondria and Takotsubo syndrome (TTS), which is closely connected with catecholamine overstimulation, poor outcomes, and changes in lipid metabolism. We investigated the changes in lipid metabolism at the level of fatty acid ß-oxidation and changes in the intracellular lipidomic spectrum. The immortalized cell line of HL-1 cardiomyocytes was used in this study as an established in vitro model of TTS. The cells were exposed to the non-selective ß-agonist isoprenaline (ISO) for acute (2 h) and prolonged (24 h) periods. We investigated the impact on mitochondrial adenosine 5'-triphosphate (ATP) production and ß-oxidation using real-time cell metabolic analysis, total lipid content, and changes in the lipidomic spectrum using high-performance liquid chromatography (HPLC) and mass spectrometry. Furthermore, modifications of selected lipid transporters were determined using real-time - polymerase chain reaction (RT-PCR) and/or Western blot techniques. By choosing this wide range of targets, we provide a detailed overview of molecular changes in lipid metabolism during catecholamine overstimulation. The present study demonstrates that acute exposure to ISO decreased ATP production by up to 42.2%, and prolonged exposure to ISO decreased ß-oxidation by 86.4%. Prolonged exposure to ISO also increased lipid accumulation by 4%. Lipid spectrum analysis of prolonged exposure to ISO showed a reduced concentration of cardioprotective and an increased concentration of lipotoxic lipid molecules during long-term exposure. Decreased lipid utilization can lead to higher intracellular lipid accumulation and the formation of lipotoxic molecules. Changes in the lipid spectrum can induce pathophysiological signaling pathways leading to cardiomyocyte remodeling or apoptosis. Thus, changes in lipid metabolism induced by excessive doses of catecholamines may cause TTS and contribute to a progression of heart failure, which is at increased risk after a TTS episode.

Supercritical fluid chromatography coupled to high-resolution tandem mass spectrometry: an innovative one-run method for the comprehensive assessment of chocolate quality and authenticity.

To assess chocolate quality and authenticity comprehensively, a combination of various analytical procedures is involved, thereby making the process time-consuming and costly. Thus, we investigated the potential of ultra-high performance supercritical fluid chromatography coupled to quadrupole-time of flight mass spectrometry (UHPSFC-QTOF-MS) as an alternative to "classic" methods. By combining hexane and aqueous extracts from sequential extraction, a single 8-min analytical run enabled us (i) to determine cocoa butter equivalents (CBEs) and milk fat content based on the detection of selected triacylglycerols, (ii) to calculate dry non-fat cocoa solids based on determined theobromine and caffeine content, and (iii) to profile contained sugars. To obtain the most comprehensive information about sample composition, the MS method comprised a full MS scan for non-target screening and several time-scheduled targeted MS/MS functions ("parallel reaction monitoring") optimized according to the possible concentration ranges of the analytes. For 40 different chocolate samples, our results and those obtained by using standard methods (LC-UV for non-fat cocoa solids, and GC-FID for CBEs) were in good agreement. Compared to the conventional approach for chocolate quality and authenticity control, the presented SFC-MS method is a fast, cost-effective, and efficient alternative, and only samples suspicious for the presence of CBE should be referred to the standard GC-FID method for exact CBE quantification. In the study, also some challenges offered by SFC-MS have been addressed.

Interlaboratory Comparison Investigations (ICIs) and External Quality Assurance Schemes (EQUASs) for human biomonitoring of perfluoroalkyl substances (PFASs) in serum as part of the quality assurance programme under HBM4EU.

Perfluoroalkyl substances (PFASs) are of very high concern due to their persistence and accumulative behaviour as well as their manifold adverse health effects. Human biomonitoring (HBM) based on the determination of PFASs in serum samples is an adequate and established strategy for exposure and risk assessment of the population. The suspected health risks associated with exposure levels in the general population call for reliable HBM data verified by Quality Assurance and Quality Control (QA/QC) measures. PFASs were among the chemicals selected as priority substances in HBM4EU, a pan-European project to harmonize and advance HBM within 30 European countries. For this purpose, the analytical comparability and accuracy of PFASs-analysing laboratories was assessed in a QA/QC programme comprising Interlaboratory Comparison Investigations (ICIs) and External Quality Assurance Schemes (EQUASs). This paper presents the evaluation process and discusses the results of four ICI/EQUAS rounds for the determination of eight perfluoroalkyl carboxylic acids and four perfluoroalkyl sulfonic acids (PFBS, PFHxS, PFHpS, PFOS) in serum. All 21 participating laboratories achieved satisfactory results for at least six of these biomarkers, although low limits of quantification (of about 0.1 µg/L) were required to quantify serum PFAS levels at general population exposure levels. The mean relative standard deviation of the participants' results (study RSDR) significantly improved from 22 % to 13 % over all PFAS biomarkers in the course of the four rounds. This QA/QC programme succeeded in establishing a network of laboratories with high analytical comparability and accuracy for the analysis of PFASs across 12 European countries.