Acylcarnitine profiling in meningiomas with different NF2 mutation status

Acylcarnitine profiling in meningiomas with different NF2 mutation status | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Acylcarnitine profiling in meningiomas with different NF2 mutation status Joanna Bogusiewicz, Jacek Furtak, Marcin Birski, Krystyna Soszyńska, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5513508/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The mutation in NF2 is the most common alteration associated with meningioma oncogenesis, and it is related to the loss of a suppressing protein called merlin. At the same time, alterations in energy production are visible in cancer cells where increased demand for energy is observed. Fatty acid oxidation could be one of the ways cancer cells obtain energy. This metabolic pathway uses the acylcarnitine shuttle system, which is responsible for the acylation of fatty acids and their transport through mitochondria. Therefore, this study aimed to profile acylcarnitines with short-, medium- and long-acyl chain length in meningiomas to assess their changes in tumors with different NF2 mutation statuses. For the analysis, solid-phase microextraction (SPME) coupled with liquid chromatography high resolution mass spectrometry (LC-HRMS) was used. The presented sampling method enables low invasive and easy collection of the analytes from the studied lesions, which can be crucial for future analysis of potential biomarkers in the surgery room. It was observed that higher levels of these analytes characterized meningiomas with NF2 mutation. Moreover, increased energy consumption and elevated levels of acylcarnitines show that these analytes can be considered as a marker of increased fatty acid oxidation in NF2 mutated cells. acylcarnitine solid-phase microextraction meningioma merlin NF2 Figures Figure 1 Figure 2 1. Introduction Meningiomas are the most common benign brain tumors. The treatment is based on the surgery; however, the tumor cannot be entirely removed or is inaccessible in some cases. Also, it should be mentioned that some percent of meningiomas can evolve into 2 or 3-grade tumors [ 1 – 3 ]. In this case, treatment is much more complex, and alternative therapies like chemotherapy or radiotherapy must be applied [ 1 – 3 ]. Therefore, basic research in the direction that enables an understanding of the relation between genetic mutations, their translation to molecular biology, and, subsequently, the impact on mechanisms behind the sudden increase of malignancy of meningiomas or resistance to particular therapy is of great importance [ 2 ]. Until now, no genetic biomarker has been used in meningioma diagnosis. Research revealed that mutation in NF2 is the most common alteration associated with meningioma oncogenesis [ 4 ]. The protein encoded by this gene is merlin, which regulates cell adhesion and signaling and impacts tumorigenesis suppression [ 5 ]. The lack of merlin correlates with a higher incidence of multiple meningiomas and schwannomas in the central nervous system and a higher risk of developing malignant lesions [ 3 , 5 ]. Stepanova et al. observed that cells with NF mutation have significantly higher fatty acid synthesis, described by the higher activity of FASN, ACC 1 and 2, and other enzymes in this metabolic pathway [ 5 ]. The authors also suspected some fatty acid oxidation alteration could be observed in samples lacking merlin. The explanation of this observation could be related to the phenomena described by Melone et al. as a “futile cycle” where two metabolic cycles going in opposite directions could be used by cancer cells. Fatty acid biosynthesis supplies appropriate fatty acid levels, while their oxidation in mitochondria provides energy for proliferating cells [ 6 ]. Intermediates that play a crucial role in fatty acid oxidation in mitochondria are acylcarnitines, esters of carnitine, and fatty acids [ 6 , 7 ]. The carnitine shuttle system evolved due to the impermeability of the mitochondrial membranes to fatty acids with long acyl chains [ 6 , 8 ]. Specialized enzymes, such as the carnitine palmitoyltransferase 1 (CPT1) and 2 (CPT2), the carnitine-acylcarnitine translocase (CACT), and the carnitine acetyltransferase (CrAT), catalyze the reactions enabling transport of acyl chains of fatty acids through mitochondrial membranes to matrix resulting in energy production as well as acyl chains catabolism [ 6 , 9 ]. Alterations in energy production are visible in cancer cells where increased demand for energy is observed [ 6 ]. Moreover, lipid metabolism is upregulated if glucose availability decreases to sustain growth and survival in unfavorable conditions [ 6 ]. Thus, the carnitine system plays an essential role in cancer metabolic plasticity. Studies on the carnitine shuttle system are usually related to assessing crucial enzyme activity, but reports on the profiling of direct acylcarnitine intermediates were also proposed [ 8 , 10 ]. However, the bottleneck of this approach is the need to homogenize studied tissue and time-consuming analysis. Therefore, methods that can cope with these problems are needed. Solid phase microextraction (SPME) was one of the methods applied for acylcarnitines analysis in brain tumors [ 8 ]. SPME is based on the interaction between sorbent coated on the small-size support (e.g., fiber with a diameter of ca. 200 µm) and analytes in the analyzed sample [ 11 ]. The probe is introduced into the tissue for a particular time, after which it is stored or proceeded to the next step: desorption of analytes into organic solution. Subsequently, samples can be analyzed using chromatography coupled with a mass spectrometer or other analytical instrumentation. Linking the information on the impaired suppressor activity of merlin in NF2 mutant meningiomas and the role of fatty acid oxidation in energy production in cancerous tumors, acylcarnitine profiling was performed to test in greater detail if the acylcarnitine profile changes are dependent on different NF2 mutation statuses. Moreover, SPME probes were applied as a sampling method due to their reported low invasiveness to the patient, low toxicity to the environment, and simplicity of sampling procedure. 2. Materials and Methods 2.1. Chemicals and materials External calibrant Pierce LTQ Velos ESI Positive Ion Calibration Solution was purchased from Thermo Scientific. Isopropanol, methanol, water, acetonitrile, and ammonium acetate were LC-MS grade and were purchased from Merck (Warsaw, Poland). SPME C18 fibers were kindly provided by Supelco (Bellefonte, PA, USA). 2.2. Biological material Brain tumors were obtained during neurosurgical procedures in the 10th Military Research Hospital and Polyclinic in Bydgoszcz. SPME sampling was conducted directly after tumor removal. Meningothelial meningiomas without mutations in AKT1, PIK3CA, TRAF, and KLF4 were selected for this study: 22 tumors with a mutation in NF2 (NF2mt) and 18 samples without this genetic alteration (NF2wt). Only first-grade tumors were included in the analysis. 2.3. Genetic testing The genetic testing of mutations in the following genes: NF2, AKT1, PIK3CA (rs104886003, rs121913273, and rs121913279), TRAF (N520C, R653Q, R641C, and K615E), and KLF4 was carried out. Analysis was performed using polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and real-time quantitative PCR (RQ-PCR). Tests were proceeded according to the manufacturer’s protocol. 2.4. Chemical biopsy (Solid-phase microextraction) protocol and LC-HRMS analysis Solid-phase microextraction probes coated with 7mm C18 sorbent were used to sample brain tumors removed during the neurosurgical procedure. The fibers were preconditioned overnight in methanol: water (1:1 v/v) solution, and then directly before sampling, they were rinsed in water. Subsequently, the probe was inserted into the tissue for 30 minutes (extraction), and after this time, it was rinsed briefly in water. Probes were stored in a freezer at − 30°C until instrumental analysis. Then, the fibers were desorbed into 150µl of isopropanol: methanol (1:1 v/v) solution using silanized inserts. Desorption was conducted for 1 hour under agitation at 850 rpm [ 12 ]. Pooled quality control (QC) and extraction blanks were also prepared [ 12 ]. The liquid chromatography-high resolution mass spectrometry (LC–HRMS) platform consisted of a Dionex UltiMate 3000 RS autosampler, a Dionex Ultimate 3000 RS pump (Thermo Fisher Scientific, Dionex, Bremen, Germany), and a Q Exactive Focus high-resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for instrumental analysis. LC analysis was conducted using 5 mM ammonium acetate in water as phase A and acetonitrile as phase B. Column: SeQuantZIC-cHILIC (3 µm 100 × 2.1 mm) was used, and the injection volume was set at 10 µL. The hydrophilic interaction chromatography (HILIC) was used in the analysis. The detailed parameters were given elsewhere [ 8 ]. The study was conducted in positive ion mode in a 100–1000 m/z scan range. Acylcarnitines were identified by matching their fragmentation patterns with spectra libraries at a mass accuracy of < 3 ppm (the presence of characteristic m/z: 85.0290 in MS/MS spectra). Full MS/dd-MS2 discovery mode was used for this purpose, and the detailed parameters of the fragmentation protocol were given elsewhere [ 8 ]. 2.5. Data processing and statistical analysis Acylcarnitine identification was performed using XCalibur software (Thermo Fisher Scientific, San Jose, CA, USA) based on m/z and characteristic fragmentation pattern. The peak areas for the obtained compounds were analyzed using MetaboAnalyst 6.0 and Statistica 13.3 PL software (StatSoft, Inc., Tulsa, Oklahoma, United States) [ 13 ]. Chemometric analysis, box-plot visualization, and receiver operating characteristic(ROC) curves were prepared. The average peak area, coefficient of variation, and the ratio of compared study groups for all analytes were calculated; the Mann–Whitney U Test was applied to compare the variables. The p-value lower than 0.05 was set as a statistical significance threshold. Finally, ChlorTox was calculated along with the recommendation given by Nowak et al. [ 14 ]. 3. Results and Discussion A set of several acylcarnitines was extracted from brain tumors using SPME fibers, and a relative standard deviation (RSD) was below 30%. Among detected analytes were short-chain acylcarnitines (SCAC): AC C2:0, AC C3:0, AC C4:0 and AC C5:0, medium-chain acylcarnitines (MCAC): AC C6:0, AC C8:0, AC C10:0, AC C10:1, AC C12:0, and long-chain acylcarnitines (LCAC): AC C14:0, AC C14:1, AC C16:0, AC C16:1, AC C18:0, AC C18:1 (Table 1 ). A similar set of analytes was extracted in a study on acylcarnitine profiling in gliomas, where SPME probes were also used for sampling [ 8 ]. Analysis was conducted in intact tissue to check if metabolite changes were observed directly in the studied tissue. This approach allows the selection of tissue-specific potential biomarkers, which, in further research, can be searched and assessed in material with easier access, e.g., blood. Table 1 The ratio of meningiomas with NF2 mutation to the tumor without this mutation was assessed using the SPME-LC-HRMS platform. AC – acylcarnitine, AUC – area under curve, FDR – False discovery rate, LCAC – long-chain acylcarnitine, MCAC – medium-chain acylcarnitine, NF2mt – NF2 mutated, NF2wt – NF2 wildtype, SCAC – short-chain acylcarnitines, RT – retention time. Acylcarnitine m/z RT Raw data NF2mt/ NF2wt ratio p-value FDR AUC SCAC AC C2:0 204.1230 13.49 3.33 < 0.05 < 0.05 0.899 AC C3:0 218.1387 11.94 2.50 < 0.05 < 0.05 0.785 AC C4:0 232.1543 10.70 2.50 < 0.05 < 0.05 0.778 AC C5:0 246.1700 9.92 2.28 0.206 0.219 0.619 MCAC AC C6:0 260.1856 9.28 2.31 < 0.05 < 0.05 0.770 AC C8:0 288.2169 8.60 2.16 < 0.05 < 0.05 0.760 AC C10:0 316.2484 8.24 2.41 < 0.05 < 0.05 0.765 AC C10:1 314.2326 8.29 1.77 0.066 0.066 0.672 AC C12:0 344.2796 7.95 2.21 < 0.05 < 0.05 0.775 LCAC AC C14:0 372.3108 7.75 1.94 < 0.05 < 0.05 0.742 AC C14:1 370.2952 7.73 2.00 < 0.05 < 0.05 0.727 AC C16:0 400.3423 7.63 1.63 0.055 0.074 0.679 AC C16:1 398.3266 7.65 2.06 < 0.05 < 0.05 0.702 AC C18:0 428.3734 7.63 1.11 0.219 0.219 0.616 AC C18:1 426.3579 7.49 1.68 0.119 0.137 0.646 A chemometric analysis using a principal component analysis (PCA) was conducted. Visualization of meningioma samples showed that NF2 mutant (NF2mt) tumors were more dispersed in the plot than NF2 wildtype (NF2wt) samples, which created a more concentrated group (Fig. 1 ). The lack of merlin, a suppressor and microtubule stabilizer protein in NFmt meningiomas, could be related to more heterogenous metabolism and energy demands, impacting the acylcarnitine profile. The presence of NF2 mutation can relate to a higher possibility of developing new foci of cancer origin or lead to tumor transformation or the occurrence of multiple tumors [ 15 ]. A trend of higher acylcarnitine levels in the samples lacking merlin (NF2mt) than in the wild type was observed. This observation corresponds to ratios of peak areas in NF2 mutant to NF2 wildtype meningiomas in Table 1 , where levels of ten out of fifteen acylcarnitines were significantly different (p < 0.05) (Fig. 2 ). Receiver operating characteristic (ROC) curves were prepared for studied analytes, and it was observed that significantly changed acylcarnitines were characterized by AUC higher than 0.7, confirming the potential of these analytes as biomarkers (Table 1 , Fig. S1). It should also be mentioned that AUC for the model built on all studied analytes was 0.719, showing that profiling of acylcarnitines can have diagnostic potential in the differentiation of tumors with different NF2 mutation statuses (Fig. S2) The lack of merlin can be related to higher malignancy and increased energy consumption, which can be observed in cancerous cells [ 16 ]. This demand could be fulfilled by changing glucose metabolism into aerobic glycolysis– Warburg effect, elevated glutamine metabolism, or changes in fatty acid oxidation [ 5 , 6 , 17 ]. Indeed, it was reported in the literature that NF2 mutant cells are characterized by higher dependence on lipid metabolism [ 5 ]. Thus, elevated acylcarnitine levels could explain increased energy consumption and fatty acid oxidation in NF2 mutated cells. It should be mentioned herein that higher levels of acylcarnitines were observed in various types of cancerous lesion glioma and breast cancer hepatocellular carcinoma in comparison to respective non-cancerous samples [ 8 , 10 , 18 – 20 ]. Results presented herein show changes in acylcarnitine levels in meningioma with different mutation NF2 status. However, it would be beneficial to enrich this research with the assessment of carnitine shuttle enzyme activity and the expression of genes responsible for their production. It would help to select the most important acylcarnitines in cancer diagnosis, especially considering LCAC alteration explained in the literature by the changes of CPT-2 activity [ 21 , 22 , 24 , 25 ]. Moreover, a method enabling fast and quantitative analysis should be introduced. The chromatographic analysis takes about half an hour to analyze one sample. Moreover, this time is even longer if sample preparation is counted. Thus, to increase the chances of clinical use of acylcarnitine analysis in meningioma diagnosis, it would be useful to optimize the method, enabling fast, quantitative, and reliable analysis of potential biomarkers. An additional advantage would be low invasiveness, as represented by the methods based on SPME. Therefore, technology, such as coated blade spray mass spectrometry (CBS) or microfluidic open interface (MOI), could be applied [ 27 ]. The CBS sampling is conducted with the probe in the shape of a sword coated with the sorbent at the tip. Then, the blade is mounted in the interface installed in the ion source. Subsequently, the drop of desorption solvent is added to the surface of the probe, and high voltage is applied. Results could be acquired in a few seconds. CBS was tested for carnitine analysis in glioma homogenate [ 28 ]. Another solution could be microfluidic open ion source MOI mass spectrometry based on the coated fiber sampling [ 29 ]. However, instead of desorption followed by instrumental analysis, the probe is put to the interface installed on the mass spectrometer. The interface consists of a chamber filled with desorption solvent. The probe is introduced to this chamber for a few seconds, during which desorption is conducted. Then, the solution with desorbed analytes is directly injected into the mass spectrometer. The combination of desorption and instrumental analysis allows a reduction of analysis time. As important as low invasiveness and the possibility of rapid analysis, introducing methods harmless to the environment can be crucial. Therefore, the objective factor, such as the ChlorTox, was calculated for the studied analytical platform. This parameter enables estimation of substance toxicity in comparison to the standard substance – chloroform [ 30 ]. It was shown that ChlorTox for solvents used for instrumental analysis is comparable with other methods presented in the literature[ 14 ]. Due to the wide application of homogenization followed by liquid-liquid extraction (LLE) in tissue analysis, the liquid-liquid extraction coupled with high-performance chromatography (LLE/HPLC) method given by Nowak et al. [ 14 , 27 ] was used as a reference. For instance, the ChlorTox for HPLC analysis was 3.36g, while in the results presented herein, it was 3.87g (Table 2 ) [ 14 ]. On the other hand, ChlorTox for sample preparation protocol was significantly different. The ChlorTox was 2.78g for the LLE-HPLC method compared to 0.21g for the SPME method [ 14 ]. This observation shows that SPME as a sample preparation method is more environment-friendly than LLE. It should be noted that ChlorTox per sample was calculated based on the number of studied samples, blanks, and QC samples. Data could also be biased due to limited information on the analytical methods for LLE/HPLC. Table 2 The calculation of the hazards of the SPME-LC-MS method in acylcarnitine analysis using HILIC chromatography and high-resolution mass spectrometry per sample. Analysis step Reagents CAS CHsub ChlorTox [g] Total ChlorTox [g] SPME Methanol 67-56-1 4.81 0.16 0.21 Isopropanol 67-63-0 3.13 0.05 Instrumental Analysis Ammonium acetate 631-61-8 0.00 0.00 3.87 Acetonitrile 75-05-8 2.25 3.87 4. Conclusions Application of SPME enabled simple profiling of a wide range of acylcarnitines in meningiomas and showed that the presence of NF2 could alter acylcarnitine profile. The loss of merlin coded by NF2 was related to a higher heterogeneity of acylcarnitine profile and increased levels of detected carnitine esters. These results suggest that alteration in the acylcarnitine system could be crucial in assessing energy usage in cancerous cells and could be potential biomarkers of neoplastic changes. However, this observation has to be confirmed by the data conducted on a bigger group of patients. Moreover, applying SPME as a sampling and sample preparation method opens new possibilities for future application and reduces environmental toxicity compared with usually used methods, such as LLE. Declarations Funding: Genetic tests were funded by the National Science Centre Poland within research grant No. 2019/33/N/ST4/00286. The National Science Centre Poland supported acylcarnitine profiling within research grant No. 2015/18/M/ST4/00059. Competing Interests: The authors declare no conflicts of interest. Data Availability: Spreadsheets with peak areas for acylcarnitines are presented in Supplementary Materials. The raw files generated during study presented herein are available from the corresponding author on reasonable request. Author Contributions: Conceptualization: J.B., M.H. and B.B.; Methodology: J.B.; Investigation: J.B., K.S., A.M., A.R., Resources: J.F., M.B., M.H. and B.B.; Data curation: J.B, Writing, original draft preparation: J.B.; Writing, review, and editing: B.B.; Visualization: J.B.; Supervision: B.B.; Project administration: J.B. and B.B.; Funding acquisition: J.B. and B.B. All authors have read and agreed to the published version of the manuscript. Ethics approval: The study was conducted in accordance with the Declaration of Helsinki and approved by the Bioethical Committee in Bydgoszcz (KB 628/2015). Consent to participate: Informed consent was obtained from all subjects involved in the study. Acknowledgments: The authors acknowledge Supelco/MilliporeSigma for kindly supplying the SPME probes. The authors would like to thank Magdalena Gaca-Tabaszewska and Paulina Szeliska for their technical support in the laboratory. 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Anal Chem 90:. https://doi.org/10.1021/acs.analchem.7b04295 Nowak PM, Bis A, Zima A (2023) ChlorTox Base – a useful source of information on popular reagents in terms of chemical hazards and greenness assessment. Green Anal Chem 6:. https://doi.org/10.1016/j.greeac.2023.100065 Additional Declarations The authors declare no competing interests. Supplementary Files Supplfile1.docx File 1: Fig. S1. Receiver operating characteristic(ROC) curves for significantly altered acylcarnitines in NF2 mutated and NF2 wildtype meningiomas and Fig. S2. Receiver operating characteristic(ROC) curve for all studied acylcarnitines in NF2 mutated and NF2 wildtype meningiomas. Supplfile2.xlsx File 2: Spreadsheets with peak areas. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5513508","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":381889370,"identity":"4b88f8ff-22ff-4e6c-8a4b-aa333b16a2f6","order_by":0,"name":"Joanna Bogusiewicz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Bogusiewicz","suffix":""},{"id":381889371,"identity":"19a75aa5-e2dd-4a64-b9bd-b49c426c4b36","order_by":1,"name":"Jacek Furtak","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jacek","middleName":"","lastName":"Furtak","suffix":""},{"id":381889372,"identity":"d6adbb17-b9c3-4e8b-b47e-2d1a686f8a26","order_by":2,"name":"Marcin Birski","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Marcin","middleName":"","lastName":"Birski","suffix":""},{"id":381889373,"identity":"9c5ce7d2-80f5-49ab-ac6b-64d2e04c8871","order_by":3,"name":"Krystyna Soszyńska","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Krystyna","middleName":"","lastName":"Soszyńska","suffix":""},{"id":381889374,"identity":"979fbc57-eea1-43b5-bd65-b350d4bd5adc","order_by":4,"name":"Anna Majdańska","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Majdańska","suffix":""},{"id":381889375,"identity":"6cd9a90f-361c-43b8-860b-f6685d846286","order_by":5,"name":"Agata Ryfa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Ryfa","suffix":""},{"id":381889376,"identity":"fda058f7-1b61-42b1-9127-c5d9b0f8064e","order_by":6,"name":"Marek Harat","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Marek","middleName":"","lastName":"Harat","suffix":""},{"id":381889377,"identity":"bbc9845d-2832-4d6a-b0ad-fb1682b9ae6d","order_by":7,"name":"Barbara Bojko","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACAwkGBsMGBhswh7GBgUGGWC1pcC08RGkBqjxMghZz6eYDhTMqzsvrNvCYbpzBcIewFss5xxIMN5y5bbjtAI/ZzQ0Mz4hw2I0cA8OHbbcTzEBaHjAcJlrLOVK1bGw7ANGygRgtYL/MOJNsuO0wW9nNGQZE+AUYYscMeyrs5M2ON2+72VNxR46gFiBgMwBTzGB3HiBGBwPzAyQOcVpGwSgYBaNgZAEA2xFBsQAIPzMAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Bojko","suffix":""}],"badges":[],"createdAt":"2024-11-24 10:49:46","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5513508/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5513508/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69832510,"identity":"aff2fd1f-25d4-4893-aff3-31b7d9bcd22a","added_by":"auto","created_at":"2024-11-25 15:45:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67468,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCA visualizes NFmt and NFwt meningiomas based on an acylcarnitine profile. A. The PCA with pooled QC, B. The PCA without pooled QC. QC- pooled quality control (red dots), NF2mt – NF2 mutated (grey dots), NF2wt – NF2 wildtype (cyan dots).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5513508/v1/fb84fad0a9d6e4601800122a.png"},{"id":69834140,"identity":"2e150232-16d8-4e95-898a-d5e3309044d6","added_by":"auto","created_at":"2024-11-25 15:53:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":82549,"visible":true,"origin":"","legend":"\u003cp\u003eBox plots representing levels of significantly altered acylcarnitines in NF2 mutated and NF2 wildtype meningiomas analyzed using SPME coupled with LC-HRMS (p\u0026lt;0.05). Plots for raw data are given. NF2mt – NF2 mutated (grey boxes), NF2wt – NF2 wildtype (cyan boxes). The black dots represent the peak areas of the selected acylcarnitine from all samples. The notch shows the 95% confidence interval around the median of each group. The yellow diamond indicates the mean concentration of each group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5513508/v1/7f8472b92f1e98d4cc79ce19.png"},{"id":69834887,"identity":"c718958b-7b2f-46bb-a838-15cfbb143e3a","added_by":"auto","created_at":"2024-11-25 16:10:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":623912,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5513508/v1/8fb06fbd-068d-496c-8bdd-4c5e6a4a9818.pdf"},{"id":69832513,"identity":"afcd088f-2b74-465c-972c-ef7c93252460","added_by":"auto","created_at":"2024-11-25 15:45:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":528065,"visible":true,"origin":"","legend":"\u003cp\u003eFile 1: Fig. S1. Receiver operating characteristic(ROC) curves for significantly altered acylcarnitines in NF2 mutated and NF2 wildtype meningiomas and Fig. S2. Receiver operating characteristic(ROC) curve for all studied acylcarnitines in NF2 mutated and NF2 wildtype meningiomas.\u003c/p\u003e","description":"","filename":"Supplfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5513508/v1/0fcc73668e1c4184c7b9de30.docx"},{"id":69832515,"identity":"51171a07-99f0-4804-ada7-d19392d88f8a","added_by":"auto","created_at":"2024-11-25 15:45:54","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":18519,"visible":true,"origin":"","legend":"\u003cp\u003eFile 2: Spreadsheets with peak areas.\u003c/p\u003e","description":"","filename":"Supplfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5513508/v1/18bfb546a9eb83514c925f42.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eAcylcarnitine profiling in meningiomas with different NF2 mutation status\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMeningiomas are the most common benign brain tumors. The treatment is based on the surgery; however, the tumor cannot be entirely removed or is inaccessible in some cases. Also, it should be mentioned that some percent of meningiomas can evolve into 2 or 3-grade tumors [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In this case, treatment is much more complex, and alternative therapies like chemotherapy or radiotherapy must be applied [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, basic research in the direction that enables an understanding of the relation between genetic mutations, their translation to molecular biology, and, subsequently, the impact on mechanisms behind the sudden increase of malignancy of meningiomas or resistance to particular therapy is of great importance [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Until now, no genetic biomarker has been used in meningioma diagnosis. Research revealed that mutation in NF2 is the most common alteration associated with meningioma oncogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The protein encoded by this gene is merlin, which regulates cell adhesion and signaling and impacts tumorigenesis suppression [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The lack of merlin correlates with a higher incidence of multiple meningiomas and schwannomas in the central nervous system and a higher risk of developing malignant lesions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Stepanova et al. observed that cells with NF mutation have significantly higher fatty acid synthesis, described by the higher activity of FASN, ACC 1 and 2, and other enzymes in this metabolic pathway [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The authors also suspected some fatty acid oxidation alteration could be observed in samples lacking merlin. The explanation of this observation could be related to the phenomena described by Melone et al. as a \u0026ldquo;futile cycle\u0026rdquo; where two metabolic cycles going in opposite directions could be used by cancer cells. Fatty acid biosynthesis supplies appropriate fatty acid levels, while their oxidation in mitochondria provides energy for proliferating cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntermediates that play a crucial role in fatty acid oxidation in mitochondria are acylcarnitines, esters of carnitine, and fatty acids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The carnitine shuttle system evolved due to the impermeability of the mitochondrial membranes to fatty acids with long acyl chains [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Specialized enzymes, such as the carnitine palmitoyltransferase 1 (CPT1) and 2 (CPT2), the carnitine-acylcarnitine translocase (CACT), and the carnitine acetyltransferase (CrAT), catalyze the reactions enabling transport of acyl chains of fatty acids through mitochondrial membranes to matrix resulting in energy production as well as acyl chains catabolism [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Alterations in energy production are visible in cancer cells where increased demand for energy is observed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Moreover, lipid metabolism is upregulated if glucose availability decreases to sustain growth and survival in unfavorable conditions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, the carnitine system plays an essential role in cancer metabolic plasticity. Studies on the carnitine shuttle system are usually related to assessing crucial enzyme activity, but reports on the profiling of direct acylcarnitine intermediates were also proposed [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the bottleneck of this approach is the need to homogenize studied tissue and time-consuming analysis. Therefore, methods that can cope with these problems are needed. Solid phase microextraction (SPME) was one of the methods applied for acylcarnitines analysis in brain tumors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSPME is based on the interaction between sorbent coated on the small-size support (e.g., fiber with a diameter of ca. 200 \u0026micro;m) and analytes in the analyzed sample [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The probe is introduced into the tissue for a particular time, after which it is stored or proceeded to the next step: desorption of analytes into organic solution. Subsequently, samples can be analyzed using chromatography coupled with a mass spectrometer or other analytical instrumentation.\u003c/p\u003e \u003cp\u003eLinking the information on the impaired suppressor activity of merlin in NF2 mutant meningiomas and the role of fatty acid oxidation in energy production in cancerous tumors, acylcarnitine profiling was performed to test in greater detail if the acylcarnitine profile changes are dependent on different NF2 mutation statuses. Moreover, SPME probes were applied as a sampling method due to their reported low invasiveness to the patient, low toxicity to the environment, and simplicity of sampling procedure.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and materials\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExternal calibrant Pierce LTQ Velos ESI Positive Ion Calibration Solution was purchased from Thermo Scientific. Isopropanol, methanol, water, acetonitrile, and ammonium acetate were LC-MS grade and were purchased from Merck (Warsaw, Poland). SPME C18 fibers were kindly provided by Supelco (Bellefonte, PA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Biological material\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBrain tumors were obtained during neurosurgical procedures in the 10th Military Research Hospital and Polyclinic in Bydgoszcz. SPME sampling was conducted directly after tumor removal. Meningothelial meningiomas without mutations in AKT1, PIK3CA, TRAF, and KLF4 were selected for this study: 22 tumors with a mutation in NF2 (NF2mt) and 18 samples without this genetic alteration (NF2wt). Only first-grade tumors were included in the analysis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Genetic testing\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe genetic testing of mutations in the following genes: NF2, AKT1, PIK3CA (rs104886003, rs121913273, and rs121913279), TRAF (N520C, R653Q, R641C, and K615E), and KLF4 was carried out. Analysis was performed using polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and real-time quantitative PCR (RQ-PCR). Tests were proceeded according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Chemical biopsy (Solid-phase microextraction) protocol and LC-HRMS analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSolid-phase microextraction probes coated with 7mm C18 sorbent were used to sample brain tumors removed during the neurosurgical procedure. The fibers were preconditioned overnight in methanol: water (1:1 v/v) solution, and then directly before sampling, they were rinsed in water. Subsequently, the probe was inserted into the tissue for 30 minutes (extraction), and after this time, it was rinsed briefly in water. Probes were stored in a freezer at \u0026minus;\u0026thinsp;30\u0026deg;C until instrumental analysis. Then, the fibers were desorbed into 150\u0026micro;l of isopropanol: methanol (1:1 v/v) solution using silanized inserts. Desorption was conducted for 1 hour under agitation at 850 rpm [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Pooled quality control (QC) and extraction blanks were also prepared [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe liquid chromatography-high resolution mass spectrometry (LC\u0026ndash;HRMS) platform consisted of a Dionex UltiMate 3000 RS autosampler, a Dionex Ultimate 3000 RS pump (Thermo Fisher Scientific, Dionex, Bremen, Germany), and a Q Exactive Focus high-resolution mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for instrumental analysis.\u003c/p\u003e \u003cp\u003eLC analysis was conducted using 5 mM ammonium acetate in water as phase A and acetonitrile as phase B. Column: SeQuantZIC-cHILIC (3 \u0026micro;m 100 \u0026times; 2.1 mm) was used, and the injection volume was set at 10 \u0026micro;L. The hydrophilic interaction chromatography (HILIC) was used in the analysis. The detailed parameters were given elsewhere [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The study was conducted in positive ion mode in a 100\u0026ndash;1000 m/z scan range. Acylcarnitines were identified by matching their fragmentation patterns with spectra libraries at a mass accuracy of \u0026lt;\u0026thinsp;3 ppm (the presence of characteristic m/z: 85.0290 in MS/MS spectra). Full MS/dd-MS2 discovery mode was used for this purpose, and the detailed parameters of the fragmentation protocol were given elsewhere [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data processing and statistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAcylcarnitine identification was performed using XCalibur software (Thermo Fisher Scientific, San Jose, CA, USA) based on m/z and characteristic fragmentation pattern. The peak areas for the obtained compounds were analyzed using MetaboAnalyst 6.0 and Statistica 13.3 PL software (StatSoft, Inc., Tulsa, Oklahoma, United States) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Chemometric analysis, box-plot visualization, and receiver operating characteristic(ROC) curves were prepared. The average peak area, coefficient of variation, and the ratio of compared study groups for all analytes were calculated; the Mann\u0026ndash;Whitney U Test was applied to compare the variables. The p-value lower than 0.05 was set as a statistical significance threshold.\u003c/p\u003e \u003cp\u003eFinally, ChlorTox was calculated along with the recommendation given by Nowak et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA set of several acylcarnitines was extracted from brain tumors using SPME fibers, and a relative standard deviation (RSD) was below 30%. Among detected analytes were short-chain acylcarnitines (SCAC): AC C2:0, AC C3:0, AC C4:0 and AC C5:0, medium-chain acylcarnitines (MCAC): AC C6:0, AC C8:0, AC C10:0, AC C10:1, AC C12:0, and long-chain acylcarnitines (LCAC): AC C14:0, AC C14:1, AC C16:0, AC C16:1, AC C18:0, AC C18:1 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A similar set of analytes was extracted in a study on acylcarnitine profiling in gliomas, where SPME probes were also used for sampling [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Analysis was conducted in intact tissue to check if metabolite changes were observed directly in the studied tissue. This approach allows the selection of tissue-specific potential biomarkers, which, in further research, can be searched and assessed in material with easier access, e.g., blood.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe ratio of meningiomas with NF2 mutation to the tumor without this mutation was assessed using the SPME-LC-HRMS platform. AC \u0026ndash; acylcarnitine, AUC \u0026ndash; area under curve, FDR \u0026ndash; False discovery rate, LCAC \u0026ndash; long-chain acylcarnitine, MCAC \u0026ndash; medium-chain acylcarnitine, NF2mt \u0026ndash; NF2 mutated, NF2wt \u0026ndash; NF2 wildtype, SCAC \u0026ndash; short-chain acylcarnitines, RT \u0026ndash; retention time.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eAcylcarnitine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em/z\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eRT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eRaw data\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNF2mt/ NF2wt ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFDR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAUC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eSCAC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C2:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e204.1230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.899\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C3:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e218.1387\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.785\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C4:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232.1543\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C5:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e246.1700\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.619\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eMCAC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C6:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e260.1856\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.770\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C8:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e288.2169\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.760\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C10:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e316.2484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.765\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C10:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e314.2326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.066\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.672\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C12:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e344.2796\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.775\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"6\"\u003e\n \u003cp\u003e\u003cstrong\u003eLCAC\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C14:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e372.3108\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.742\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C14:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e370.2952\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.727\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C16:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e400.3423\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.055\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.679\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C16:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e398.3266\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;\u0026thinsp;0.05\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.702\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C18:0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e428.3734\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.219\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.616\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAC C18:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e426.3579\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.119\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.646\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eA chemometric analysis using a principal component analysis (PCA) was conducted. Visualization of meningioma samples showed that NF2 mutant (NF2mt) tumors were more dispersed in the plot than NF2 wildtype (NF2wt) samples, which created a more concentrated group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The lack of merlin, a suppressor and microtubule stabilizer protein in NFmt meningiomas, could be related to more heterogenous metabolism and energy demands, impacting the acylcarnitine profile. The presence of NF2 mutation can relate to a higher possibility of developing new foci of cancer origin or lead to tumor transformation or the occurrence of multiple tumors [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eA trend of higher acylcarnitine levels in the samples lacking merlin (NF2mt) than in the wild type was observed. This observation corresponds to ratios of peak areas in NF2 mutant to NF2 wildtype meningiomas in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, where levels of ten out of fifteen acylcarnitines were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Receiver operating characteristic (ROC) curves were prepared for studied analytes, and it was observed that significantly changed acylcarnitines were characterized by AUC higher than 0.7, confirming the potential of these analytes as biomarkers (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. S1). It should also be mentioned that AUC for the model built on all studied analytes was 0.719, showing that profiling of acylcarnitines can have diagnostic potential in the differentiation of tumors with different NF2 mutation statuses (Fig. S2)\u003c/p\u003e\n\u003cp\u003eThe lack of merlin can be related to higher malignancy and increased energy consumption, which can be observed in cancerous cells [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. This demand could be fulfilled by changing glucose metabolism into aerobic glycolysis\u0026ndash; Warburg effect, elevated glutamine metabolism, or changes in fatty acid oxidation [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. Indeed, it was reported in the literature that NF2 mutant cells are characterized by higher dependence on lipid metabolism [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e]. Thus, elevated acylcarnitine levels could explain increased energy consumption and fatty acid oxidation in NF2 mutated cells. It should be mentioned herein that higher levels of acylcarnitines were observed in various types of cancerous lesion glioma and breast cancer hepatocellular carcinoma in comparison to respective non-cancerous samples [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eResults presented herein show changes in acylcarnitine levels in meningioma with different mutation NF2 status. However, it would be beneficial to enrich this research with the assessment of carnitine shuttle enzyme activity and the expression of genes responsible for their production. It would help to select the most important acylcarnitines in cancer diagnosis, especially considering LCAC alteration explained in the literature by the changes of CPT-2 activity [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, a method enabling fast and quantitative analysis should be introduced. The chromatographic analysis takes about half an hour to analyze one sample. Moreover, this time is even longer if sample preparation is counted. Thus, to increase the chances of clinical use of acylcarnitine analysis in meningioma diagnosis, it would be useful to optimize the method, enabling fast, quantitative, and reliable analysis of potential biomarkers.\u003c/p\u003e\n\u003cp\u003eAn additional advantage would be low invasiveness, as represented by the methods based on SPME. Therefore, technology, such as coated blade spray mass spectrometry (CBS) or microfluidic open interface (MOI), could be applied [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The CBS sampling is conducted with the probe in the shape of a sword coated with the sorbent at the tip. Then, the blade is mounted in the interface installed in the ion source. Subsequently, the drop of desorption solvent is added to the surface of the probe, and high voltage is applied. Results could be acquired in a few seconds. CBS was tested for carnitine analysis in glioma homogenate [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. Another solution could be microfluidic open ion source MOI mass spectrometry based on the coated fiber sampling [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, instead of desorption followed by instrumental analysis, the probe is put to the interface installed on the mass spectrometer. The interface consists of a chamber filled with desorption solvent. The probe is introduced to this chamber for a few seconds, during which desorption is conducted. Then, the solution with desorbed analytes is directly injected into the mass spectrometer. The combination of desorption and instrumental analysis allows a reduction of analysis time.\u003c/p\u003e\n\u003cp\u003eAs important as low invasiveness and the possibility of rapid analysis, introducing methods harmless to the environment can be crucial. Therefore, the objective factor, such as the ChlorTox, was calculated for the studied analytical platform. This parameter enables estimation of substance toxicity in comparison to the standard substance \u0026ndash; chloroform [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. It was shown that ChlorTox for solvents used for instrumental analysis is comparable with other methods presented in the literature[\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Due to the wide application of homogenization followed by liquid-liquid extraction (LLE) in tissue analysis, the liquid-liquid extraction coupled with high-performance chromatography (LLE/HPLC) method given by Nowak et al. [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] was used as a reference. For instance, the ChlorTox for HPLC analysis was 3.36g, while in the results presented herein, it was 3.87g (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. On the other hand, ChlorTox for sample preparation protocol was significantly different. The ChlorTox was 2.78g for the LLE-HPLC method compared to 0.21g for the SPME method [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. This observation shows that SPME as a sample preparation method is more environment-friendly than LLE. It should be noted that ChlorTox per sample was calculated based on the number of studied samples, blanks, and QC samples. Data could also be biased due to limited information on the analytical methods for LLE/HPLC.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe calculation of the hazards of the SPME-LC-MS method in acylcarnitine analysis using HILIC chromatography and high-resolution mass spectrometry per sample.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAnalysis step\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReagents\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCAS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCHsub\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eChlorTox [g]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal ChlorTox [g]\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSPME\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67-56-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIsopropanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67-63-0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eInstrumental Analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAmmonium acetate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e631-61-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cp\u003e3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAcetonitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75-05-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eApplication of SPME enabled simple profiling of a wide range of acylcarnitines in meningiomas and showed that the presence of NF2 could alter acylcarnitine profile. The loss of merlin coded by NF2 was related to a higher heterogeneity of acylcarnitine profile and increased levels of detected carnitine esters. These results suggest that alteration in the acylcarnitine system could be crucial in assessing energy usage in cancerous cells and could be potential biomarkers of neoplastic changes. However, this observation has to be confirmed by the data conducted on a bigger group of patients. Moreover, applying SPME as a sampling and sample preparation method opens new possibilities for future application and reduces environmental toxicity compared with usually used methods, such as LLE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Genetic tests were funded by the National Science Centre Poland within research grant No. 2019/33/N/ST4/00286. The National Science Centre Poland supported acylcarnitine profiling within research grant No. 2015/18/M/ST4/00059.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e Spreadsheets with peak areas for acylcarnitines are presented in Supplementary Materials. The raw files generated during study presented herein are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization: J.B., M.H. and B.B.; Methodology: J.B.; Investigation: J.B., K.S., A.M., A.R., Resources: J.F., M.B., M.H. and B.B.; Data curation: J.B, Writing, original draft preparation: J.B.; Writing, review, and editing: B.B.; Visualization: J.B.; Supervision: B.B.;\u0026nbsp;\u003cbr\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e J.B. and B.B.; Funding acquisition: J.B. and B.B. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e The study was conducted in accordance with the Declaration of Helsinki and approved by the Bioethical Committee in Bydgoszcz (KB 628/2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors acknowledge Supelco/MilliporeSigma for kindly supplying the SPME probes. The authors would like to thank Magdalena Gaca-Tabaszewska and Paulina Szeliska for their technical support in the laboratory. The authors would like to acknowledge Paulina Zofia Goryńska, Krzysztof Goryński and Karol Jaroch for their help with sampling the first cases.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGoldbrunner R, Minniti G, Preusser M, et al (2016) EANO guidelines for the diagnosis and treatment of meningiomas. Lancet Oncol 17:e383\u0026ndash;e391. https://doi.org/10.1016/S1470-2045(16)30321-7\u003c/li\u003e\n\u003cli\u003eNowosielski M, Galldiks N, Iglseder S, et al (2017) Diagnostic challenges in meningioma. Neuro Oncol 19:. https://doi.org/10.1093/neuonc/nox101\u003c/li\u003e\n\u003cli\u003eGupta S, Bi WL, Dunn IF (2018) Medical management of meningioma in the era of precision medicine. Neurosurg Focus 44:E3. https://doi.org/10.3171/2018.1.FOCUS17754\u003c/li\u003e\n\u003cli\u003eGhalavand MA, Asghari A, Farhadi M, et al (2023) The genetic landscape and possible therapeutics of neurofibromatosis type 2. 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Molecules 27:1\u0026ndash;13. https://doi.org/10.3390/molecules27072251\u003c/li\u003e\n\u003cli\u003eTascon M, Alam MN, G\u0026oacute;mez-R\u0026iacute;os GA, Pawliszyn J (2018) Development of a Microfluidic Open Interface with Flow Isolated Desorption Volume for the Direct Coupling of SPME Devices to Mass Spectrometry. Anal Chem 90:. https://doi.org/10.1021/acs.analchem.7b04295\u003c/li\u003e\n\u003cli\u003eNowak PM, Bis A, Zima A (2023) ChlorTox Base \u0026ndash; a useful source of information on popular reagents in terms of chemical hazards and greenness assessment. Green Anal Chem 6:. https://doi.org/10.1016/j.greeac.2023.100065\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"379b8138-7f54-4ae0-ad6e-3096c2c14431","identifier":"10.13039/501100004281","name":"Narodowe Centrum Nauki","awardNumber":"2019/33/N/ST4/00286","order_by":0},{"identity":"90605f9b-055e-4ce2-9ae6-cf864dad04f1","identifier":"10.13039/501100004281","name":"Narodowe Centrum Nauki","awardNumber":"2015/18/M/ST4/00059","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Nicolaus Copernicus University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"acylcarnitine, solid-phase microextraction, meningioma, merlin, NF2","lastPublishedDoi":"10.21203/rs.3.rs-5513508/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5513508/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mutation in NF2 is the most common alteration associated with meningioma oncogenesis, and it is related to the loss of a suppressing protein called merlin. At the same time, alterations in energy production are visible in cancer cells where increased demand for energy is observed. Fatty acid oxidation could be one of the ways cancer cells obtain energy. This metabolic pathway uses the acylcarnitine shuttle system, which is responsible for the acylation of fatty acids and their transport through mitochondria. Therefore, this study aimed to profile acylcarnitines with short-, medium- and long-acyl chain length in meningiomas to assess their changes in tumors with different NF2 mutation statuses. For the analysis, solid-phase microextraction (SPME) coupled with liquid chromatography high resolution mass spectrometry (LC-HRMS) was used. The presented sampling method enables low invasive and easy collection of the analytes from the studied lesions, which can be crucial for future analysis of potential biomarkers in the surgery room. It was observed that higher levels of these analytes characterized meningiomas with NF2 mutation. Moreover, increased energy consumption and elevated levels of acylcarnitines show that these analytes can be considered as a marker of increased fatty acid oxidation in NF2 mutated cells.\u003c/p\u003e","manuscriptTitle":"Acylcarnitine profiling in meningiomas with different NF2 mutation status","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 15:45:49","doi":"10.21203/rs.3.rs-5513508/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"095f6529-7c26-4bc6-9111-c176bf5250e5","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T15:45:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-25 15:45:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5513508","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5513508","identity":"rs-5513508","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}