TY - JOUR A1 - Hauffe, Robert A1 - Rath, Michaela A1 - Agyapong, Wilson A1 - Jonas, Wenke A1 - Vogel, Heike A1 - Schulz, Tim Julius A1 - Schwarz, Maria A1 - Kipp, Anna Patricia A1 - Blüher, Matthias A1 - Kleinridders, André T1 - Obesity Hinders the Protective Effect of Selenite Supplementation on Insulin Signaling JF - Antioxidants N2 - The intake of high-fat diets (HFDs) containing large amounts of saturated long-chain fatty acids leads to obesity, oxidative stress, inflammation, and insulin resistance. The trace element selenium, as a crucial part of antioxidative selenoproteins, can protect against the development of diet-induced insulin resistance in white adipose tissue (WAT) by increasing glutathione peroxidase 3 (GPx3) and insulin receptor (IR) expression. Whether selenite (Se) can attenuate insulin resistance in established lipotoxic and obese conditions is unclear. We confirm that GPX3 mRNA expression in adipose tissue correlates with BMI in humans. Cultivating 3T3-L1 pre-adipocytes in palmitate-containing medium followed by Se treatment attenuates insulin resistance with enhanced GPx3 and IR expression and adipocyte differentiation. However, feeding obese mice a selenium-enriched high-fat diet (SRHFD) only resulted in a modest increase in overall selenoprotein gene expression in WAT in mice with unaltered body weight development, glucose tolerance, and insulin resistance. While Se supplementation improved adipocyte morphology, it did not alter WAT insulin sensitivity. However, mice fed a SRHFD exhibited increased insulin content in the pancreas. Overall, while selenite protects against palmitate-induced insulin resistance in vitro, obesity impedes the effect of selenite on insulin action and adipose tissue metabolism in vivo. KW - selenite KW - insulin KW - adipose tissue KW - obesity KW - insulin resistance Y1 - 2022 U6 - https://doi.org/10.3390/antiox11050862 SN - 2076-3921 VL - 11 SP - 1 EP - 16 PB - MDPI CY - Basel, Schweiz ET - 5 ER - TY - THES A1 - Schell, Mareike T1 - Investigating the effect of Lactobacillus rhamnosus GG on emotional behavior in diet-induced obese C57BL/6N mice T1 - Untersuchung der Wirkung von Lactobacillus rhamnosus GG bei Störungen des emotionalen Verhaltens in einem Mausmodell Diät-induzierter Adipositas N2 - The prevalence of depression and anxiety is increased in obese patients compared to healthy humans, which is partially due to a shared pathogenesis, including insulin resistance and inflammation. These factors are also linked to intestinal dysbiosis. Additionally, the chronic consumption of diets rich in saturated fats results in body weight gain, hormonal resistances and unfavorable changes in the microbiome composition. The intake of Lactobacilli has already been shown to improve dysbiosis along with metabolism and mood. Yet, the beneficial role and the underlying mechanism of Lactobacillus rhamnosus GG (LGG) to improve emotional behavior in established diet-induced obese conditions are, so far, unknown. To characterize the role of LGG in diet-induced obesity, female and male C57BL/6N mice were fed a semi-synthetic low-fat diet (LFD, 10 % kcal from fat) or a conventional high-fat diet (HFD, 45 % kcal from fat) for initial 6 weeks, which was followed by daily oral gavage of vehicle or 1x10^8 CFU of LGG until the end of the experiment. Mice were subjected to basic metabolic and extensive behavioral phenotyping, with a focus on emotional behavior. Moreover, composition of cecal gut microbiome, metabolomic profile in plasma and cerebrospinal fluid was investigated and followed by molecular analyses. Both HFD-feeding and LGG application resulted in sex-specific differences. While LGG prevented the increase of plasma insulin, adrenal gland weight and hyperactivity in diet-induced obese female mice, there was no regulation of anxiodepressive-like behavior. In contrast, metabolism of male mice did not benefit from LGG application, but strikingly, LGG decreased specifically depressive-like behavior in the Mousetail Suspension Test which was confirmed by the Splash Test characterizing motivation for ’self-care’. The microbiome analysis in male mice revealed that HFD-feeding, but not LGG application, altered cecal microbiome composition, indicating a direct effect of LGG on behavioral regulation. However, in female mice, both HFD-feeding and LGG application resulted in changes of microbiome composition, which presumably affected metabolism. Moreover, as diet-induced obese female mice unexpectedly did not exhibit anxiodepressive-like behavior, follow-up analyses were conducted in male mice. Here, HFD-feeding significantly altered abundance of plasma lipids whereas LGG decreased branched chain amino acids which associated with improved emotional behavior. In nucleus accumbens (NAcc) and VTA/SN, which belong to the dopaminergic system, LGG restored HFD-induced decrease of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, on gene expression level. Lastly, transcriptome analysis in the NAcc identified gene expression of cholecystokinin as a potential mediator of the effect of LGG on HFD-induced emotional alterations. In summary, this thesis revealed the beneficial effects of LGG application on emotional alterations in established diet-induced obesity. Furthermore, both HFD-feeding and LGG treatment exhibited sex-specific effects, resulting in metabolic improvements in female mice while LGG application mitigated depressive-like behavior in obese male mice along with a molecular signature of restored dopamine synthesis and neuropeptide signaling. N2 - n adipösen Patienten liegt eine erhöhte Prävalenz von Depressionen und Angsterkrankungen vor. Dies liegt unter anderem an einer gemeinsamen Pathogenese, der eine Insulinresistenz sowie ein chronischer Entzündungszustand zugrunde liegen. Diese Faktoren sind mit einer intestinalen Dysbiose assoziiert, die auch durch eine Fehlernährung, beispielsweise mit einer fettreichen Diät, hervorgerufen werden kann. Es konnte bereits gezeigt werden, dass die Aufnahme von Laktobazillen nicht nur eine Dysbiose und den Stoffwechsel verbessert, sondern sich auch positiv auf das Gemüt auswirken kann. Ob jedoch Lactobacillus rhamnosus GG in der Lage ist, in einem Zustand der etablierten ernährungsbedingten Fettleibigkeit das emotionale Verhalten zu verbessern und welche Mechanismen zugrunde liegen, ist noch ungeklärt. Um die Rolle von LGG bei ernährungsbedingter Fettleibigkeit zu charakterisieren, wurden weibliche und männliche C57BL/6N Mäuse mit einer semi-synthetischen Niedrigfettdiät (LFD, 10 % kcal aus Fett) oder einer konventionellen Hochfettdiät (HFD, 45 % kcal aus Fett) für die ersten 6 Wochen gefüttert, um den Zustand einer Adipositas zu etablieren. Anschließend haben die Mäuse eine tägliche perorale Applikation eines Vehikels oder 1x10^8 KBE LGG bis zum Versuchsende erhalten. Die Mäuse wurden einer allgemeinen metabolischen Charakterisierung und einer umfassenden Verhaltensphänotypisierung unterzogen, die Aufschlüsse über das emotionale Verhalten liefern sollen. Darüber hinaus wurde die Zusammensetzung des Darmmikrobioms bestimmt, im Plasma und in der Zerebrospinalflüssigkeit das Metabolitprofil untersucht und durch molekulare Analysen ergänzt. Sowohl die HFD-Fütterung als auch die LGG-Applikation führten zu geschlechtsspezifischen Unterschieden. Während LGG den diätinduzierten Anstieg von Plasmainsulin, ein erhöhtes Nebennierengewicht und Hyperaktivität in weiblichen Mäusen verhinderte, wurde das emotionale Verhalten nicht reguliert. Im Gegensatz dazu profitierte der Stoffwechsel männlicher Mäuse nicht von der LGG-Anwendung, jedoch war LGG in der Lage, spezifisch das depressiv-ähnliches Verhalten zu verbessern, was durch eine Analyse des zielgerichteten Verhaltens bestätigt wurde. Die Mikrobiomanalyse ergab, dass die Diät, jedoch nicht LGG, die Zusammensetzung des Darmmikrobioms in männlichen Mäusen verändert, was auf eine direkte Rolle von LGG in der Verhaltensregulation hindeutet. Im Vergleich dazu war das Darmmikrobiom in weiblichen Mäusen durch die Diät als auch durch LGG verändert, was zu den positiven Veränderungen der Stoffwechselparameter geführt haben könnte. Da weibliche Mäuse weder durch die HFD-Fütterung noch durch die LGG-Gabe einen Effekt auf emotionales Verhalten aufwiesen, wurden die Folgeanalysen bei männlichen Mäusen durchgeführt. Während die HFD-Fütterung das Vorkommen von Plasmalipiden veränderte, lagen aufgrund der LGG-Gabe verzweigtkettige Aminosäuren verringert vor, was mit einem verbessertem emotionalen Verhalten assoziierte. In den dopaminergen Gehirnregionen Nucleus Accumbens (NAcc) und VTA/SN revertierte LGG die HFD-induzierte Reduktion der Tyrosinhydroxylase Genexpression, des geschwindigkeitsbegrenzenden Enzyms in der Dopaminsynthese. Abschließend wurde eine Transkriptomanalyse mittels RNA Sequencing durchgeführt, welche die Genexpression von Cholezystokinin im NAcc als potenzieller Mediator in der Wirkung von LGG bei HFD-induzierten emotionalen Veränderungen identifizierte. Zusammenfassend konnten in dieser Arbeit die positiven Auswirkungen der LGG-Gabe auf emotionales Verhalten bei etablierter ernährungsbedingter Fettleibigkeit gezeigt werden.. Sowohl die HFD-Fütterung als auch die LGG-Gabe führten zu geschlechtsspezifischen Effekten, was zu Stoffwechselverbesserungen bei weiblichen Mäusen führte, während die LGG-Gabe das depressiv-ähnliche Verhalten bei männlichen Mäusen abschwächte. Zudem wurden auf Genexpressionsebene Tyrosinhydroxylase und Cholezystokinin identifiziert, die potentiell den Effekt von LGG auf das emotionale Verhalten in einem Modell etablierter ernährungsbedingter Fettleibigkeit vermitteln. KW - obesity KW - insulin resistance KW - probiotics KW - lactobacillus KW - depression KW - emotionality Y1 - 2022 ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Püschel, Gerhard Paul T1 - Discrimination of the activity of low-affinity wild-type and high-affinity mutant recombinant BoNT/B by a SIMA cell-based reporter release assay JF - Toxins N2 - Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma–based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data. KW - cell-based assay KW - genetically modified BoNT KW - BoNT/B uptake Y1 - 2022 U6 - https://doi.org/10.3390/toxins14010065 SN - 2072-6651 VL - 14 SP - 1 EP - 11 PB - MDPI CY - Basel, Schweiz ET - 1 ER - TY - JOUR A1 - Püschel, Gerhard A1 - Klauder, Julia A1 - Henkel-Oberländer, Janin T1 - Macrophages, low-grade inflammation, insulin resistance and hyperinsulinemia BT - A mutual ambiguous relationship in the development of metabolic diseases JF - Journal of Clinical Medicine : open access journal N2 - Metabolic derangement with poor glycemic control accompanying overweight and obesity is associated with chronic low-grade inflammation and hyperinsulinemia. Macrophages, which present a very heterogeneous population of cells, play a key role in the maintenance of normal tissue homeostasis, but functional alterations in the resident macrophage pool as well as newly recruited monocyte-derived macrophages are important drivers in the development of low-grade inflammation. While metabolic dysfunction, insulin resistance and tissue damage may trigger or advance pro-inflammatory responses in macrophages, the inflammation itself contributes to the development of insulin resistance and the resulting hyperinsulinemia. Macrophages express insulin receptors whose downstream signaling networks share a number of knots with the signaling pathways of pattern recognition and cytokine receptors, which shape macrophage polarity. The shared knots allow insulin to enhance or attenuate both pro-inflammatory and anti-inflammatory macrophage responses. This supposedly physiological function may be impaired by hyperinsulinemia or insulin resistance in macrophages. This review discusses the mutual ambiguous relationship of low-grade inflammation, insulin resistance, hyperinsulinemia and the insulin-dependent modulation of macrophage activity with a focus on adipose tissue and liver. KW - NAFLD/MAFLD KW - type 2 diabetes KW - obesity KW - vicious cycle KW - TLR signaling KW - M1/M2 differentiation KW - Akt pathway Y1 - 2022 U6 - https://doi.org/10.3390/jcm11154358 SN - 2077-0383 VL - 11 IS - 15 SP - 1 EP - 30 PB - MDPI CY - Basel, Schweiz ER - TY - JOUR A1 - Fechner, Carolin A1 - Hackethal, Christin A1 - Höpfner, Tobias A1 - Dietrich, Jessica A1 - Bloch, Dorit A1 - Lindtner, Oliver A1 - Sarvan, Irmela T1 - Results of the BfR MEAL Study BT - in Germany, mercury is mostly contained in fish and seafood while cadmium, lead, and nickel are present in a broad spectrum of foods JF - Food chemistry: X N2 - The BfR MEAL Study provides representative levels of substances in foods consumed in Germany. Mercury, cadmium, lead, and nickel are contaminants present in foods introduced by environmental and industrial processes. Levels of these elements were investigated in 356 foods. Foods were purchased representatively, prepared as consumed and pooled with similar foods before analysis. Highest mean levels of mercury were determined in fish and seafood, while high levels of cadmium, lead, and nickel were present in cocoa products and legumes, nuts, oilseeds, and spices. The sampling by region, season, and production type showed minor differences in element levels for specific foods, however no tendency over all foods or for some food groups was apparent. The data on mercury, cadmium, lead, and nickel provide a comprehensive basis for chronic dietary exposure assessment of the population in Germany. All levels found were below regulated maximum levels. KW - Total diet study KW - BfR MEAL Study KW - Metals KW - Contaminants KW - Unprepared and KW - prepared foods KW - Regionality KW - Seasonality KW - Organic and conventional type of production Y1 - 2022 U6 - https://doi.org/10.1016/j.fochx.2022.100326 SN - 2590-1575 VL - 14 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Knoche, Lisa A1 - Lisec, Jan A1 - Koch, Matthias T1 - Analysis of electrochemical and liver microsomal transformation products of lasalocid by LC/HRMS JF - Rapid communications in mass spectrometry : RCM N2 - Rationale: Lasalocid (LAS), an ionophore, is used in cattle and poultry farming as feed additive for its antibiotic and growth-promoting properties. Literature on transformation products (TP) resulting from LAS degradation is limited. So far, only hydroxylation is found to occur as the metabolic reaction during the LAS degradation. To investigate potential TPs of LAS, we used electrochemistry (EC) and liver microsome (LM) assays to synthesize TPs, which were identified using liquid chromatography high-resolution mass spectrometry (LC/HRMS). Methods: Electrochemically produced TPs were analyzed online by direct coupling of the electrochemical cell to the electrospray ionization (ESI) source of a Sciex Triple-TOF high resolution mass spectrometer. Then, EC-treated LAS solution was collected and analyzed offline using LC/HRMS to confirm stable TPs and improve their annotation with a chemical structure due to informative MS/MS spectra. In a complementary approach, TPs formed by rat and human microsomal incubation were investigated using LC/HRMS. The resulting data were used to investigate LAS modification reactions and elucidate the chemical structure of obtained TPs. Results: The online measurements identified a broad variety of TPs, resulting from modification reactions like (de-)hydrogenation, hydration, methylation, oxidation as well as adduct formation with methanol. We consistently observed different ion complexations of LAS and LAS-TPs (Na+; 2Na(+) K+; NaNH4+; KNH4+). Two stable methylated EC-TPs were found, structurally annotated, and assigned to a likely modification reaction. Using LM incubation, seven TPs were formed, mostly by oxidation/hydroxylation. After the identification of LM-TPs as Na+-complexes, we identified LM-TPs as K+-complexes. Conclusion: We identified and characterized TPs of LAS using EC- and LM-based methods. Moreover, we found different ion complexes of LAS-based TPs. This knowledge, especially the different ion complexes, may help elucidate the metabolic and environmental degradation pathways of LAS. Y1 - 2022 U6 - https://doi.org/10.1002/rcm.9349 SN - 0951-4198 SN - 1097-0231 VL - 36 IS - 18 PB - Wiley CY - New York, NY ER - TY - JOUR A1 - Schiborn, Catarina A1 - Schulze, Matthias Bernd T1 - Precision prognostics for the development of complications in diabetes JF - Diabetologia : journal of the European Association for the Study of Diabetes (EASD) N2 - Individuals with diabetes face higher risks for macro- and microvascular complications than their non-diabetic counterparts. The concept of precision medicine in diabetes aims to optimise treatment decisions for individual patients to reduce the risk of major diabetic complications, including cardiovascular outcomes, retinopathy, nephropathy, neuropathy and overall mortality. In this context, prognostic models can be used to estimate an individual's risk for relevant complications based on individual risk profiles. This review aims to place the concept of prediction modelling into the context of precision prognostics. As opposed to identification of diabetes subsets, the development of prediction models, including the selection of predictors based on their longitudinal association with the outcome of interest and their discriminatory ability, allows estimation of an individual's absolute risk of complications. As a consequence, such models provide information about potential patient subgroups and their treatment needs. This review provides insight into the methodological issues specifically related to the development and validation of prediction models for diabetes complications. We summarise existing prediction models for macro- and microvascular complications, commonly included predictors, and examples of available validation studies. The review also discusses the potential of non-classical risk markers and omics-based predictors. Finally, it gives insight into the requirements and challenges related to the clinical applications and implementation of developed predictions models to optimise medical decision making. KW - Cardiovascular diseases KW - Complications in diabetes KW - Macrovascular KW - complications KW - Microvascular complications KW - Personalised medicine KW - Precision medicine KW - Precision prognostics KW - Review KW - Risk prediction KW - Risk KW - scores Y1 - 2022 U6 - https://doi.org/10.1007/s00125-022-05731-4 SN - 0012-186X SN - 1432-0428 PB - Springer CY - New York ER - TY - JOUR A1 - Knoche, Lisa A1 - Lisec, Jan A1 - Schwerdtle, Tanja A1 - Koch, Matthias T1 - LC-HRMS-Based identification of transformation products of the drug salinomycin generated by electrochemistry and liver microsome JF - Antibiotics N2 - The drug salinomycin (SAL) is a polyether antibiotic and used in veterinary medicine as coccidiostat and growth promoter. Recently, SAL was suggested as a potential anticancer drug. However, transformation products (TPs) resulting from metabolic and environmental degradation of SAL are incompletely known and structural information is missing. In this study, we therefore systematically investigated the formation and identification of SAL derived TPs using electrochemistry (EC) in an electrochemical reactor and rat and human liver microsome incubation (RLM and HLM) as TP generating methods. Liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) was applied to determine accurate masses in a suspected target analysis to identify TPs and to deduce occurring modification reactions of derived TPs. A total of 14 new, structurally different TPs were found (two EC-TPs, five RLM-TPs, and 11 HLM-TPs). The main modification reactions are decarbonylation for EC-TPs and oxidation (hydroxylation) for RLM/HLM-TPs. Of particular interest are potassium-based TPs identified after liver microsome incubation because these might have been overlooked or declared as oxidated sodium adducts in previous, non-HRMS-based studies due to the small mass difference between K and O + Na of 21 mDa. The MS fragmentation pattern of TPs was used to predict the position of identified modifications in the SAL molecule. The obtained knowledge regarding transformation reactions and novel TPs of SAL will contribute to elucidate SAL-metabolites with regards to structural prediction. KW - salinomycin KW - ionophore antibiotics KW - transformation product KW - electrochemistry KW - rat KW - human liver microsomes KW - HRMS Y1 - 2022 U6 - https://doi.org/10.3390/antibiotics11020155 SN - 2079-6382 VL - 11 IS - 2 PB - MDPI CY - Basel ER - TY - JOUR A1 - Aga-Barfknecht, Heja A1 - Soultoukis, George A. A1 - Stadion, Mandy A1 - Garcia-Carrizo, Francisco A1 - Jähnert, Markus A1 - Gottmann, Pascal A1 - Vogel, Heike A1 - Schulz, Tim Julius A1 - Schürmann, Annette T1 - Distinct adipogenic and fibrogenic differentiation capacities of mesenchymal stromal cells from pancreas and white adipose tissue JF - International journal of molecular sciences N2 - Pancreatic steatosis associates with beta-cell failure and may participate in the development of type-2-diabetes. Our previous studies have shown that diabetes-susceptible mice accumulate more adipocytes in the pancreas than diabetes-resistant mice. In addition, we have demonstrated that the co-culture of pancreatic islets and adipocytes affect insulin secretion. The aim of this current study was to elucidate if and to what extent pancreas-resident mesenchymal stromal cells (MSCs) with adipogenic progenitor potential differ from the corresponding stromal-type cells of the inguinal white adipose tissue (iWAT). miRNA (miRNome) and mRNA expression (transcriptome) analyses of MSCs isolated by flow cytometry of both tissues revealed 121 differentially expressed miRNAs and 1227 differentially expressed genes (DEGs). Target prediction analysis estimated 510 DEGs to be regulated by 58 differentially expressed miRNAs. Pathway analyses of DEGs and miRNA target genes showed unique transcriptional and miRNA signatures in pancreas (pMSCs) and iWAT MSCs (iwatMSCs), for instance fibrogenic and adipogenic differentiation, respectively. Accordingly, iwatMSCs revealed a higher adipogenic lineage commitment, whereas pMSCs showed an elevated fibrogenesis. As a low degree of adipogenesis was also observed in pMSCs of diabetes-susceptible mice, we conclude that the development of pancreatic steatosis has to be induced by other factors not related to cell-autonomous transcriptomic changes and miRNA-based signals. KW - MSCs KW - fatty pancreas KW - WAT KW - lineage commitment KW - transcriptomics KW - miRNAs Y1 - 2022 U6 - https://doi.org/10.3390/ijms23042108 SN - 1422-0067 VL - 23 IS - 4 PB - Molecular Diversity Preservation International CY - Basel ER - TY - JOUR A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Waldbach Braga, Tess A1 - Rackiewicz, Michal A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.) JF - Processes : open access journal N2 - Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples. KW - wheat KW - α-amylase/trypsin inhibitors KW - fractionation KW - purification KW - reversed-phase chromatography KW - ion-exchange chromatography KW - design of experiment KW - LC–MS/MS KW - MALDI-TOF-MS Y1 - 2022 U6 - https://doi.org/10.3390/pr10020259 SN - 2227-9717 VL - 10 SP - 1 EP - 18 PB - MDPI CY - Basel, Schweiz ET - 2 ER -