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Traumatic brain injury (TBI) is often complicated by long-lasting disabilities, including headache, fatigue, insomnia, hyperactivity, and cognitive deficits. In a previous study in mice, we showed that persistent non-goal-directed hyperactivity is a characteristic post-TBI behavior that was associated with low levels of endocannabinoids in the perilesional cortex. We now analyzed lipidome patterns in the brain and plasma in TBI versus sham mice in association with key behavioral parameters and endocannabinoids. Lipidome profiles in the plasma and subcortical ipsilateral and contralateral brain were astonishingly equal in sham and TBI mice, but the ipsilateral perilesional cortex revealed a strong increase in neutral lipids represented by 30 species of triacylglycerols (TGs) of different chain lengths and saturation. The accumulation of TG was localized predominantly to perilesional border cells as revealed by Oil Red O staining. In addition, hexosylceramides (HexCer) and phosphatidylethanolamines (PE and ether-linked PE-O) were reduced. They are precursors of gangliosides and endocannabinoids, respectively. High TG, low HexCer, and low PE/PE-O showed a linear association with non-goal-directed nighttime hyperactivity but not with the loss of avoidance memory. The analyses suggest that TG overload and HexCer and PE deficiencies contributed to behavioral dimensions of post-TBI psychopathology.
Objective: Skin and soft tissue infections (SSTI) are a commonly known entity of diseases associated with difficult treatment procedures. The current gold standard when there is a rapidly progressing infection of soft tissues with a risk of sepsis is radical surgical debridement accompanied by systemic antibiotic therapy. In clinical settings, local antibiotics alone or formulated within carrier material are commonly used alongside this therapy regimen. One possibility of local antibiotic application is the fixation of colistin with fibrin glue spray. It is not yet sufficiently researched how the local antibiotic concentrations remain as high as possible over time.
Methods: We conducted an animal study including 29 male Wistar rats inducing sterile back sores reaching the muscle fascia. We sprayed only colistin, simultaneously or consecutively, with fibrin glue in different groups in order to measure the tissue concentration of the antibiotic applied locally.
Results: After liquid chromatography and quadrupole mass spectrometry analysis, it could be demonstrated that in comparison to the colistin group, tissue concentrations of colistin stayed significantly higher in the wound tissue when it was fixed with fibrin glue. This was observed in both groups, the simultaneous as well as in the consecutively fibrin glue sprayed groups after colistin application.
Conclusion: The fixation of colistin with the fibrin-glue-spray technique as a carrier for local antibiotic therapy is an easy and inexpensive method and shows promising potential for the treatment of SSTI.
Progranulin deficiency in mice is associated with deregulations of the scavenger receptor signaling of CD36/SCARB3 in immune disease models, and CD36 is a dominant receptor in taste bud cells in the tongue and contributes to the sensation of dietary fats. Progranulin-deficient mice (Grn−/−) are moderately overweight during middle age. We therefore asked if there was a connection between progranulin/CD36 in the tongue and fat taste preferences. By using unbiased behavioral analyses in IntelliCages and Phenomaster cages we showed that progranulin-deficient mice (Grn−/−) developed a strong preference of fat taste in the form of 2% milk over 0.3% milk, and for diluted MCTs versus tap water. The fat preference in the 7d-IntelliCage observation period caused an increase of 10% in the body weight of Grn−/− mice, which did not occur in the wildtype controls. CD36 expression in taste buds was reduced in Grn−/− mice at RNA and histology levels. There were no differences in the plasma or tongue lipids of various classes including sphingolipids, ceramides and endocannabinoids. The data suggest that progranulin deficiency leads to a lower expression of CD36 in the tongue resulting in a stronger urge for fatty taste and fatty nutrition.
Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.
Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the leading causes of liver disease worldwide. To identify disease-specific pathomechanisms, we analyzed the lipidome, metabolome and immune cell recruitment in livers in both diseases. Mice harboring ASH or NASH had comparable disease severities regarding mortality rate, neurological behavior, expression of fibrosis marker and albumin levels. Lipid droplet size was higher in NASH than ASH and qualitative differences in the lipidome were mainly based on incorporation of diet-specific fatty acids into triglycerides, phosphatidylcholines and lysophosphatidylcholines. Metabolomic analysis showed downregulated nucleoside levels in both models. Here, the corresponding uremic metabolites were only upregulated in NASH suggesting stronger cellular senescence, which was supported by lower antioxidant levels in NASH as compared to ASH. While altered urea cycle metabolites suggest increased nitric oxide synthesis in both models, in ASH, this depended on increased L-homoarginine levels indicating a cardiovascular response mechanism. Interestingly, only in NASH were the levels of tryptophan and its anti-inflammatory metabolite kynurenine upregulated. Fittingly, high-content immunohistochemistry showed a decreased macrophage recruitment and an increased polarization towards M2-like macrophages in NASH. In conclusion, with comparable disease severity in both models, higher lipid storage, oxidative stress and tryptophan/kynurenine levels were seen in NASH, leading to distinct immune responses.
Endocannabinoids (ECs) are potent lipid mediators with high physiological relevance. They are involved in a wide variety of diseases like depression or multiple sclerosis and are closely connected to metabolic parameters in humans. Therefore, their suitability as a biomarker in different (patho-)physiological conditions is discussed intensively and predominantly investigated by analyzing systemic concentrations in easily accessible matrices like blood. Carefully designed pre-analytical sample handling is of major importance for high-quality data, but harmonization is not achieved yet. Whole blood is either processed to serum or plasma before the onset of analytical workflows and while knowledge about pre-analytical challenges in plasma handling is thorough they were not systematically investigated for serum.
Therefore, the ECs AEA and 2-AG, and closely related EC-like substances 1-AG, DHEA, and PEA were examined by LC-MS/MS in serum samples of nine healthy volunteers employing different pre-analytical sample handling protocols, including prolonged coagulation, and storage after centrifugation at room temperature (RT) or on ice. Furthermore, all analytes were also assessed in plasma samples obtained from the same individuals at the same time points to investigate the comparability between those two blood-based matrices regarding obtained concentrations and their 2-AG/1-AG ratio.
This study shows that ECs and EC-like substances in serum samples were significantly higher than in plasma and are especially prone to ex vivo changes during initial and prolonged storage for coagulation at RT. Storage on ice after centrifugation is less critical. However, storage at RT further increases 1-AG and 2-AG concentrations, while also lowering the already reduced 2-AG/1-AG ratio due to isomerization. Thus, avoidance of prolonged processing at RT can increase data quality if serum as the matrix of choice is unavoidable. However, serum preparation in itself is expected to initiate changes of physiological concentrations as standard precautionary measures like fast and cooled processing can only be utilized by using plasma, which should be the preferred matrix for analyses of ECs and EC-like substances.
The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.
Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects
(2023)
Mass spectrometry focusing on small endogenous molecules has become an integral part of biomarker discovery in the pursuit of an in-depth understanding of the pathophysiology of various diseases, ultimately enabling the application of personalized medicine. While LC-MS methods allow researchers to gather vast amounts of data from hundreds or thousands of samples, the successful execution of a study as part of clinical research also requires knowledge transfer with clinicians, involvement of data scientists, and interactions with various stakeholders.
The initial planning phase of a clinical research project involves specifying the scope and design, and engaging relevant experts from different fields. Enrolling subjects and designing trials rely largely on the overall objective of the study and epidemiological considerations, while proper pre-analytical sample handling has immediate implications on the quality of analytical data. Subsequent LC-MS measurements may be conducted in a targeted, semi-targeted, or non-targeted manner, resulting in datasets of varying size and accuracy. Data processing further enhances the quality of data and is a prerequisite for in-silico analysis. Nowadays, the evaluation of such complex datasets relies on a mix of classical statistics and machine learning applications, in combination with other tools, such as pathway analysis and gene set enrichment. Finally, results must be validated before biomarkers can be used as prognostic or diagnostic decision-making tools. Throughout the study, quality control measures should be employed to enhance the reliability of data and increase confidence in the results.
The aim of this graphical review is to provide an overview of the steps to be taken when conducting an LC-MS-based clinical research project to search for small molecule biomarkers.
Introduction: Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) are central lipid mediators of the endocannabinoid system. They are highly relevant due to their involvement in a wide variety of inflammatory, metabolic or malign diseases. Further elucidation of their modes of action and use as biomarkers in an easily accessible matrix, like blood, is restricted by their susceptibility to deviations during blood sampling and physiological co-dependences, which results in high variability of reported concentrations in low ng/mL ranges.
Objectives: The objective of this review is the identification of critical parameters during the pre-analytical phase and proposal of minimum requirements for reliable determination of endocannabinoids (ECs) in blood samples.
Methods: Reported physiological processes influencing the EC concentrations were put into context with published pre-analytical research and stability data from bioanalytical method validation.
Results: The cause for variability in EC concentrations is versatile. In part, they are caused by inter-individual factors like sex, metabolic status and/or diurnal changes. Nevertheless, enzymatic activity in freshly drawn blood samples is the main reason for changing concentrations of AEA and 2-AG, besides additional non-enzymatic isomerization of the latter.
Conclusion: Blood samples for EC analyses require immediate processing at low temperatures (>0 °C) to maintain sample integrity. Standardization of the respective blood tube or anti-coagulant, sampling time point, applied centrifugal force and complete processing time can further decrease variability caused by sample handling. Nevertheless, extensive characterization of study participants is needed to reduce distortion of clinical data caused by co-variables and facilitate research on the endocannabinoid system.
We recently described that monoacylglycerol lipase (MGL) is present in the tumor microenvironment (TME), increasing tumor growth. In this study we compare the implications of MGL deficiency in the TME in different tumor types.
We show that subcutaneous injection of KP (KrasLSL-G12D/p53fl/fl, mouse lung adenocarcinoma) or B16-F10 cells (mouse melanoma) induced tumor growth in MGL wild type (WT) and knockout (KO) mice. MGL deficiency in the TME attenuated the growth of KP cell tumors whereas tumors from B16-F10 cells increased in size. Opposite immune cell profiles were detected between the two tumor types in MGL KO mice. In line with their anti-tumorigenic function, the number of CD8+ effector T cells and eosinophils increased in KP cell tumors of MGL KO vs. WT mice whereas their presence was reduced in B16-F10 cell tumors of MGL KO mice. Differences were seen in lipid profiles between the investigated tumor types. 2-arachidonoylglycerol (2-AG) content significantly increased in KP, but not B16-F10 cell tumors of MGL KO vs. WT mice while other endocannabinoid-related lipids remained unchanged. However, profiles of phospho- and lysophospholipids, sphingomyelins and fatty acids in KP cell tumors were clearly distinct to those measured in B16-F10 cell tumors.
Our data indicate that TME-localized MGL impacts tumor growth, as well as levels of 2-AG and other lipids in a tumor specific manner.