Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.

Mapping the metabolic responses to oxaliplatin-based chemotherapy with in vivo spatiotemporal metabolomics.

Adjuvant chemotherapy improves the survival outlook for patients undergoing operations for lung metastases caused by colorectal cancer (CRC). However, a multidisciplinary approach that evaluates several factors related to patient and tumor characteristics is necessary for managing chemotherapy treatment in metastatic CRC patients with lung disease, as such factors dictate the timing and drug regimen, which may affect treatment response and prognosis. In this study, we explore the potential of spatial metabolomics for evaluating metabolic phenotypes and therapy outcomes during the local delivery of the anticancer drug, oxaliplatin, to the lung. 12 male Yorkshire pigs underwent a 3 h left lung in vivo lung perfusion (IVLP) with various doses of oxaliplatin (7.5, 10, 20, 40, and 80 mg/L), which were administered to the perfusion circuit reservoir as a bolus. Biocompatible solid-phase microextraction (SPME) microprobes were combined with global metabolite profiling to obtain spatiotemporal information about the activity of the drug, determine toxic doses that exceed therapeutic efficacy, and conduct a mechanistic exploration of associated lung injury. Mild and subclinical lung injury was observed at 40 mg/L of oxaliplatin, and significant compromise of the hemodynamic lung function was found at 80 mg/L. This result was associated with massive alterations in metabolic patterns of lung tissue and perfusate, resulting in a total of 139 discriminant compounds. Uncontrolled inflammatory response, abnormalities in energy metabolism, and mitochondrial dysfunction next to accelerated kynurenine and aldosterone production were recognized as distinct features of dysregulated metabolipidome. Spatial pharmacometabolomics may be a promising tool for identifying pathological responses to chemotherapy.

The metabolic fate of oxaliplatin in the biological milieu investigated during in vivo lung perfusion using a unique miniaturized sampling approach based on solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

Adjuvant chemotherapy after pulmonary metastasectomy for colorectal cancer may reduce recurrence and improve survival rates; however, the benefits of this treatment are limited by the significant side effects that accompany it. The development of a novel in vivo lung perfusion (IVLP) platform would permit the localized delivery of high doses of chemotherapeutic drugs to target residual micrometastatic disease. Nonetheless, it is critical to continuously monitor the levels of such drugs during IVLP administration, as lung injury can occur if tissue concentrations are not maintained within the therapeutic window. This paper presents a simple chemical-biopsy approach based on sampling with a small nitinol wire coated with a sorbent of biocompatible morphology and evaluates its applicability for the near-real-time in vivo determination of oxaliplatin (OxPt) in a 72-h porcine IVLP survival model. To this end, the pigs underwent a 3-h left lung IVLP with 3 doses of the tested drug (5, 7.5, and 40 mg/L), which were administered to the perfusion circuit reservoir as a bolus after a full perfusion flow had been established. Along with OxPt levels, the biocompatible solid-phase microextraction (SPME) probes were employed to profile other low-molecular-weight compounds to provide spatial and temporal information about the toxicity of chemotherapy or lung injury. The resultant measurements revealed a rather heterogeneous distribution of OxPt (over the course of IVLP) in the two sampled sections of the lung. In most cases, the OxPt concentration in the lung tissue peaked during the second hour of IVLP, with this trend being more evident in the upper section. In turn, OxPt in supernatant samples represented ∼25% of the entire drug after the first hour of perfusion, which may be attributable to the binding of OxPt to albumin, its sequestration into erythrocytes, or its rapid nonenzymatic biotransformation. Additionally, the Bio-SPME probes also facilitated the extraction of various endogenous molecules for the purpose of screening biochemical pathways affected during IVLP (i.e., lipid and amino acid metabolism, steroidogenesis, or purine metabolism). Overall, the results of this study demonstrate that the minimally invasive SPME-based sampling approach presented in this work can serve as (pre)clinical and precise bedside medical tool.

Dynamic Metabolic Changes During Prolonged Ex Situ Heart Perfusion Are Associated With Myocardial Functional Decline.

Ex situ heart perfusion (ESHP) was developed to preserve and evaluate donated hearts in a perfused beating state. However, myocardial function declines during ESHP, which limits the duration of perfusion and the potential to expand the donor pool. In this research, we combine a novel, minimally-invasive sampling approach with comparative global metabolite profiling to evaluate changes in the metabolomic patterns associated with declines in myocardial function during ESHP. Biocompatible solid-phase microextraction (SPME) microprobes serving as chemical biopsy were used to sample heart tissue and perfusate in a translational porcine ESHP model and a small cohort of clinical cases. In addition, six core-needle biopsies of the left ventricular wall were collected to compare the performance of our SPME sampling method against that of traditional tissue-collection. Our state-of-the-art metabolomics platform allowed us to identify a large number of significantly altered metabolites and lipid species that presented comparable profile of alterations to conventional biopsies. However, significant discrepancies in the pool of identified analytes using two sampling methods (SPME vs. biopsy) were also identified concerning mainly compounds susceptible to dynamic biotransformation and most likely being a result of low-invasive nature of SPME. Overall, our results revealed striking metabolic alterations during prolonged 8h-ESHP associated with uncontrolled inflammation not counterbalanced by resolution, endothelial injury, accelerated mitochondrial oxidative stress, the disruption of mitochondrial bioenergetics, and the accumulation of harmful lipid species. In conclusion, the combination of perfusion parameters and metabolomics can uncover various mechanisms of organ injury and recovery, which can help differentiate between donor hearts that are transplantable from those that should be discarded.

Simultaneous quantification of selected glycosaminoglycans by butanolysis-based derivatization and LC-SRM/MS analysis for assessing glycocalyx disruption in vitro and in vivo.

Glycosaminoglycans (GAGs) constitute the main building blocks of the endothelial glycocalyx (GLX), and disruption of GLX initiates and promotes endothelial dysfunction. Here, we aimed to develop a novel, specific and accurate LC-SRM/MS-based method for glycosaminoglycans (GAGs) profiling. The method involved butanolysis derivatization to facilitate GAG-specific disaccharide generation and its subsequent retention in LC-reversed-phase mode followed by mass spectrometric detection performed in positive ion-selected reaction monitoring (SRM) mode. GAG contents were measured in media of endothelial cells (EA.hy926) subjected to various GAG-degrading enzymes, as well as in murine plasma and urine in apolipoprotein E/low-density lipoprotein receptor-deficient (ApoE/LDLR -/-) mice and age-matched wild-type C57BL/6 mice. Alternatively, GLX disruption was verified by atomic force microscopy (AFM)-based analysis of GLX thickness. The proposed assay to quantify GAG-specific disaccharides presented high sensitivity for each of the analytes (LLOQ: 0.05-0.1 µg/mL) as well as accuracy and precision (86.8-114.9% and 2.0-14.3%, respectively). In medium of EA.hy926 cells subjected to GAG-degrading enzymes various GAG-specific disaccharides indicating the degradation of keratan sulphate (KS), heparan sulphate (HS), chondroitin sulphate (CHS) or hyaluronan (HA) were detected as predicted based on the characteristics of individual enzyme activity. In turn, AFM-based assessment of GLX thickness was reduced to a similar extent by all single enzyme treatments, whereas the most prominent reduction of GLX thickness was detected following the enzyme mixture. Plasma measurements of GAGs revealed age- and hypercholesterolemia-dependent decrease in GAGs concentration. In summary, a novel LC-SRM/MS-based method for GAG profiling was proposed that may inform on GLX status in cell culture for both in vitro and in vivo conditions.

SPME-LC/MS-based serum metabolomic phenotyping for distinguishing ovarian cancer histologic subtypes: a pilot study.

Epithelial ovarian cancer (EOC) is the most common cause of death from gynecological cancer. The outcomes of EOC are complicated, as it is often diagnosed late and comprises several heterogenous subtypes. As such, upfront treatment can be highly challenging. Although many significant advances in EOC management have been made over the past several decades, further work must be done to develop early detection tools capable of distinguishing between the various EOC subtypes. In this paper, we present a sophisticated analytical pipeline based on solid-phase microextraction (SPME) and three orthogonal LC/MS acquisition modes that facilitates the comprehensive mapping of a wide range of analytes in serum samples from patients with EOC. PLS-DA multivariate analysis of the metabolomic data was able to provide clear discrimination between all four main EOC subtypes: serous, endometrioid, clear cell, and mucinous carcinomas. The prognostic performance of discriminative metabolites and lipids was confirmed via multivariate receiver operating characteristic (ROC) analysis (AUC value > 88% with 20 features). Further pathway analysis using the top 57 dysregulated metabolic features showed distinct differences in amino acid, lipid, and steroids metabolism among the four EOC subtypes. Thus, metabolomic profiling can serve as a powerful tool for complementing histology in classifying EOC subtypes.

High Throughput Procedure for Comparative Analysis of In Vivo Cardiac Glucose or Amino Acids Use in Cardiovascular Pathologies and Pharmacological Treatments.

The heart is characterized by the prominent flexibility of its energy metabolism and is able to use diverse carbon substrates, including carbohydrates and amino acids. Cardiac substrate preference could have a major impact on the progress of cardiac pathologies. However, the majority of methods to investigate changes in substrates' use in cardiac metabolism in vivo are complex and not suitable for high throughput testing necessary to understand and reverse these pathologies. Thus, this study aimed to develop a simple method that would allow for the analysis of cardiac metabolic substrate use. The developed methods involved the subcutaneous injection of stable 13C isotopomers of glucose, valine, or leucine with mass spectrometric analysis for the investigation of its entry into cardiac metabolic pathways that were deducted from 13C alanine and glutamate enrichments in heart extracts. The procedures were validated by confirming the known effects of treatments that modify glucose, free fatty acids, and amino acid metabolism. Furthermore, we studied changes in the energy metabolism of CD73 knock-out mice to demonstrate the potential of our methods in experimental research. The methods created allowed for fast estimation of cardiac glucose and amino acid use in mice and had the potential for high-throughput analysis of changes in pathology and after pharmacological treatments.

Physical Activity and Inhibition of ACE Additively Modulate ACE/ACE-2 Balance in Heart Failure in Mice.

Angiotensin-converting enzyme inhibition (ACE-I) and physical activity favorably modulate the ACE/ACE-2 balance. However, it is not clear whether physical activity and ACE-I could synergistically modulate ACE/ACE-2 balance in the course of heart failure (HF). Here, we studied the effects of combined spontaneous physical activity and ACE-I-based treatment on angiotensin (Ang) pattern and cardiac function in a mouse model of HF (Tgαq*44). Tgαq*44 mice with advanced HF (at the age of 12 months) were running spontaneously in a running wheel (exercise training group, ExT) and/or were treated with ACE inhibitor (ACE-I, perindopril, 10 mg/kg) for 2 months. Angiotensin profile was characterized by an LC-MS/MS-based method. The cardiac performance was assessed in vivo by MRI. Ang-(1-7)/Ang II ratio in both plasma and the aorta was significantly higher in the combined treatment group than the ACE-I group or ExT alone, suggesting the additive favorable effects on ACE-2/Ang-(1-7) and ACE/Ang II axes' balance induced by a combination of ACE-I with ExT. The basal cardiac performance did not differ among the experimental groups of Tgαq*44 mice. We demonstrated additive changes in ACE/ACE-2 balance in both plasma and the aorta by spontaneous physical activity and ACE-I treatment in Tgαq*44 mice. However, these changes did not result in an improvement of failing heart function most likely because the disease was at the end-stage. Ang-(1-7)/Ang II balance represents a valuable biochemical end point for monitoring therapeutic intervention outcome in heart failure.

ROS Modulating Effects of Lingonberry (Vaccinium vitis-idaea L.) Polyphenols on Obese Adipocyte Hypertrophy and Vascular Endothelial Dysfunction.

Oxidative stress and dysregulated adipocytokine secretion accompanying hypertrophied adipose tissue induce chronic inflammation, which leads to vascular endothelial dysfunction. The present study investigated the ability of anthocyanin (ACN) and non-anthocyanin polyphenol (PP) fractions from lingonberry fruit to mitigate adipose tissue hypertrophy and endothelial dysfunction using 3T3-L1 adipocytes and human umbilical vein endothelial cells (HUVECs). This study showed that the PP fraction decreased intracellular ROS generation in hypertrophied adipocytes by enhancing antioxidant enzyme expression (SOD2) and inhibiting oxidant enzyme expression (NOX4, iNOS). Moreover, PP and ACN fractions reduced triglyceride content in adipocytes accompanied by downregulation of the expression of lipogenic genes such as aP2, FAS, and DAGT1. Treatment with both fractions modulated the mRNA expression and protein secretion of key adipokines in hypertrophied adipocytes. Expression and secretion of leptin and adiponectin were, respectively, down- and upregulated. Furthermore, PP and ACN fractions alleviated the inflammatory response in TNF-α-induced HUVECs by inhibiting the expression of pro-inflammatory genes (IL-6, IL-1ß) and adhesion molecules (VCAM-1, ICAM-1, SELE). The obtained results suggest that consuming polyphenol-rich lingonberry fruit may help prevent and treat obesity and endothelial dysfunction due to their antioxidant and anti-inflammatory actions.

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion.

Development of a novel in vivo lung perfusion (IVLP) procedure allows localized delivery of high-dose doxorubicin (DOX) for targeting residual micrometastatic disease in the lungs. However, DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window. A small dimension nitinol wire coated with a sorbent of biocompatible morphology (Bio-SPME) has been clinically evaluated for in vivo lung tissue extraction and determination of DOX and its key metabolites. The in vivo Bio-SPME-IVLP experiments were performed on pig model over various (150 and 225 mg/m2) drug doses, and during human clinical trial. Two patients with metastatic osteosarcoma were treated with a single 5 and 7 µg/mL (respectively) dose of DOX during a 3-h IVLP. In both pig and human cases, DOX tissue levels presented similar trends during IVLP. Human lung tissue concentrations of drug ranged between 15 and 293 µg/g over the course of the IVLP procedure. In addition to DOX levels, Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening, providing information about lung status during drug administration. Real-time monitoring of DOX levels in the lungs can be performed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach. Bio-SPME also extracted various endogenous molecules, thus providing a real-time snapshot of the physiology of the cells, which might assist in the tailoring of personalized treatment strategy.

Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry.

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.

Multi-omic signatures of atherogenic dyslipidaemia: pre-clinical target identification and validation in humans.

BACKGROUND: Dyslipidaemia is a major risk factor for atherosclerosis and cardiovascular diseases. The molecular mechanisms that translate dyslipidaemia into atherogenesis and reliable markers of its progression are yet to be fully elucidated. To address this issue, we conducted a comprehensive metabolomic and proteomic analysis in an experimental model of dyslipidaemia and in patients with familial hypercholesterolemia (FH). METHODS: Liquid chromatography/mass spectrometry (LC/MS) and immunoassays were used to find out blood alterations at metabolite and protein levels in dyslipidaemic ApoE-/-/LDLR-/- mice and in FH patients to evaluate their human relevance. RESULTS: We identified 15 metabolites (inhibitors and substrates of nitric oxide synthase (NOS), low-molecular-weight antioxidants (glutamine, taurine), homocysteine, methionine, 1-methylnicotinamide, alanine and hydroxyproline) and 9 proteins (C-reactive protein, proprotein convertase subtilisin/kexin type 9, apolipoprotein C-III, soluble intercellular adhesion molecule-1, angiotensinogen, paraoxonase-1, fetuin-B, vitamin K-dependent protein S and biglycan) that differentiated FH patients from healthy controls. Most of these changes were consistently found in dyslipidaemic mice and were further amplified if mice were fed an atherogenic (Western or low-carbohydrate, high-protein) diet. CONCLUSIONS: The alterations highlighted the involvement of an immune-inflammatory response system, oxidative stress, hyper-coagulation and impairment in the vascular function/regenerative capacity in response to dyslipidaemia that may also be directly engaged in development of atherosclerosis. Our study further identified potential biomarkers for an increased risk of atherosclerosis that may aid in clinical diagnosis or in the personalized treatment.

Enhanced cardiac hypoxic injury in atherogenic dyslipidaemia results from alterations in the energy metabolism pattern.

OBJECTIVE: Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury. METHODS: Dyslipidaemic apolipoprotein E and low-density lipoprotein receptor (ApoE/LDLR) double knockout (ApoE-/-/LDLR-/-) and wild-type C57BL/6 (WT) mice aged six months old were studied. Cardiac injury under reduced oxygen supply was evaluated by 5 min exposure to 5% oxygen in the breathing air under electrocardiogram (ECG) recording and with the assessment of troponin I release. To address the mechanisms LC/MS was used to analyse the cardiac proteome pattern or in vivo metabolism of stable isotope-labelled substrates and HPLC was applied to measure concentrations of cardiac high-energy phosphates. Furthermore, the effect of blocking fatty acid use with ranolazine on the substrate preference and cardiac hypoxic damage was studied in ApoE-/-/LDLR-/- mice. RESULTS: Hypoxia induced profound changes in ECG ST-segment and troponin I leakage in ApoE-/-/LDLR-/- mice but not in WT mice. The evaluation of the cardiac proteomic pattern revealed that ApoE-/-/LDLR-/- as compared with WT mice were characterised by coordinated increased expression of mitochondrial proteins, including enzymes of fatty acids' and branched-chain amino acids' oxidation, accompanied by decreased expression levels of glycolytic enzymes. These findings correlated with in vivo analysis, revealing a reduction in the entry of glucose and enhanced entry of leucine into the cardiac Krebs cycle, with the cardiac high-energy phosphates pool maintained. These changes were accompanied by the activation of molecular targets controlling mitochondrial metabolism. Ranolazine reversed the oxidative metabolic shift in ApoE-/-/LDLR-/- mice and reduced cardiac damage induced by hypoxia. CONCLUSIONS: We suggest a novel mechanism for myocardial injury in dyslipidaemia that is consequent to an increased reliance on oxidative metabolism in the heart. The alterations in the metabolic pattern that we identified constitute an adaptive mechanism that facilitates maintenance of metabolic equilibrium and cardiac function under normoxia. However, this adaptation could account for myocardial injury even in a mild reduction of oxygen supply.

A model to assess acute and delayed lung toxicity of oxaliplatin during in vivo lung perfusion.

OBJECTIVES: To determine the dose-limiting toxicity of oxaliplatin chemotherapy delivered by in vivo lung perfusion (IVLP). To allow assessment of subacute toxicities, we aimed to develop a 72-hour porcine IVLP survival model. METHODS: In total, 12 Yorkshire male pigs were used. Left lung IVLP was performed for 3 hours. At 72 hours postoperatively, computed tomography imaging of the lungs was performed before the pigs were killed. Lung physiology, airway dynamics, gross appearance, and histology were assessed before and during IVLP, at reperfusion, and when the pigs were euthanized. An accelerated titration dose-escalation study design was employed whereby oxaliplatin doses were sequentially doubled provided no clinically significant toxicity was observed, defined as an arterial partial pressure of oxygen to fraction of inspired oxygen ratio <300 mm Hg or severe acute lung injury on biopsy. RESULTS: After an initial training phase, no mortality or adverse events related to the procedure were observed. There was no lung injury observed at the time of IVLP for any case. At sacrifice, clinically significant lung injury was observed at 80 mg/L oxaliplatin, with an arterial partial pressure of oxygen to fraction of inspired oxygen ratio of 112 mm Hg. Mild and subclinical lung injury was observed at 40 mg/L, with this dose being repeated to confirm safety. CONCLUSIONS: A stable and reproducible porcine 3-day IVLP survival model was established that will allow toxicity assessment of agents delivered by IVLP. Oxaliplatin delivered by IVLP showed delayed-onset toxicity that was not apparent at the time of reperfusion, with a maximal-tolerated dose of 40 mg/L. This information will inform initiation of a clinical trial examining IVLP delivery of oxaliplatin at our institution.

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion / 药物分析学报

Development of a novel in vivo lung perfusion(IVLP)procedure allows localized delivery of high-dose doxorubicin(DOX)for targeting residual micrometastatic disease in the lungs.However,DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window.A small dimension nitinol wire coated with a sorbent of biocompatible morphology(Bio-SPME)has been clinically evaluated for in vivo lung tissue extraction and determina-tion of DOX and its key metabolites.The in vivo Bio-SPME-IVLP experiments were performed on pig model over various(150 and 225 mg/m2)drug doses,and during human clinical trial.Two patients with metastatic osteosarcoma were treated with a single 5 and 7 μg/mL(respectively)dose of DOX during a 3-h IVLP.In both pig and human cases,DOX tissue levels presented similar trends during IVLP.Human lung tissue concentrations of drug ranged between 15 and 293 μg/g over the course of the IVLP procedure.In addition to DOX levels,Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening,providing information about lung status during drug administration.Real-time monitoring of DOX levels in the lungs can be per-formed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach.Bio-SPME also extracted various endogenous molecules,thus providing a real-time snapshot of the physi-ology of the cells,which might assist in the tailoring of personalized treatment strategy.

Comprehensive Investigation of Metabolic Changes Occurring in the Rat Brain Hippocampus after Fluoxetine Administration Using Two Complementary In Vivo Techniques: Solid Phase Microextraction and Microdialysis.

Fluoxetine is among the most prescribed antidepressant drugs worldwide. Nevertheless, limited information is known about its definitive mechanism. Although in vivo examinations performed directly in related brain structures can provide more realistic, and therefore more insightful, knowledge regarding the mechanisms and efficacy of this drug, only a few techniques are applicable for in vivo monitoring of metabolic alterations in the brain following an inducement. Among them, solid phase microextraction (SPME) and microdialysis (MD) have emerged as ideal in vivo tools for extraction of information from biosystems. In this investigation, we scrutinized the capabilities of SPME and MD to detect ongoing changes in the brain following acute fluoxetine administration. Sequential in vivo samples were collected simultaneously from male rats' hippocampi using SPME and MD before drug administration in order to establish a baseline; then samples were collected again following fluoxetine administration for an investigation of small molecule alterations. Our results indicate that MD provides more comprehensive information for polar compounds, while SPME provides superior information with respect to lipids and other medium level polar molecules. Interestingly, in the lipidomic investigation, all dysregulated features were found to be membrane lipids and associated compounds. Moreover, in the metabolomic investigations, dysregulation of hippocampal metabolite levels associated with fatty acid transportation and purine metabolisms were among the most notable findings. Overall, our evaluation of the obtained data corroborates that, when used in tandem, SPME and MD are capable of providing comprehensive information regarding the effect of fluoxetine in targeted brain structures and further elucidating this drug's mechanisms of action in the brain.

Metabolic profile of fish muscle tissue changes with sampling method, storage strategy and time.

Unstable tissue components (metabolites) are not easily captured and evaluated by traditional metabolomics methods. In this study, a comprehensive investigation of various sampling methods and storage conditions on the metabolomic profile of fish muscle was performed based on in vivo and ex vivo sampling. The GlobalStd algorithm and structure/reaction directed analysis under a linear mixed model were used to investigate the distinctive influences of these factors on the metabolomic profiles of fish tissue obtained via untargeted LC-MS analysis. Immediate analysis of samples yielded different metabolomic profiles compared to that of stored samples. Storage time was found to affect the metabolomic profile in a complex way, whereas storage temperature was shown to not substantially change this pattern. At the reaction level, metabolites involved in homologous series with butylation were shown stable during storage. Overall, our findings demonstrate that immediate instrumental analysis after in vivo solid phase microextraction (SPME) sampling and a reverse time series experimental design should be the preferred approaches for metabolomic profiling if unstable compounds are of interest.

Investigation of Early Death-Induced Changes in Rat Brain by Solid Phase Microextraction via Untargeted High Resolution Mass Spectrometry: In Vivo versus Postmortem Comparative Study.

Analysis of brain samples obtained postmortem remains a standard approach in neuroscience, despite often being suboptimal for inferring roles of small molecules in the pathophysiology of brain diseases. Sample collection and preservation further hinders conclusive interpretation of biomarker analysis in autopsy samples. We investigate purely death-induced changes affecting rat hippocampus in the first hour of postmortem interval (PMI) by means of untargeted liquid chromatography-mass spectrometry-based metabolomics. The unique possibility of sampling the same brain area of each animal both in vivo and postmortem was enabled by employing solid phase microextraction (SPME) probes. Four millimeter probes coated with mixed mode extraction phase were used to sample awake, freely roaming animals, with 2 more sampling events performed after death. Significant changes in brain neurochemistry were found to occur as soon as 30 min after death, further progressing with increasing PMI, evidenced by relative changes in levels of metabolites and lipids. These included species from several distinct groups, which can be classified as engaged in energy metabolism-related processes, signal transduction, neurotransmission, or inflammatory response. Additionally, we perform thorough analysis of interindividual variability in response to death, which provides insights into how this aspect can obscure conclusions drawn from an untargeted study at single metabolite and pathway level. The results suggest high demand for systematic studies examining the PMI time course with in vivo sampling as a starting point to eliminate artifacts in the form of neurochemical changes assumed to occur in vivo.

In†Vivo Solid-Phase Microextraction for Sampling of Oxylipins in Brain of Awake, Moving Rats.

Oxylipins are key lipid mediators of important brain processes, including pain, sleep, oxidative stress, and inflammation. For the first time, an in-depth profile of up to 52 oxylipins can be obtained from the brains of awake moving animals using in vivo solid-phase microextraction (SPME) chemical biopsy tool in combination with liquid chromatography-high resolution mass spectrometry. Among these, 23 oxylipins are detectable in the majority of healthy wildtype samples. This new approach successfully eliminates the changes in oxylipin concentrations routinely observed during the analysis of post-mortem samples, allows time-course monitoring of their concentrations with high spatial resolution in specific brain regions of interest, and can be performed using the same experimental set-up as in vivo microdialysis (MD) thus providing a new and exciting tool in neuroscience and drug discovery.

Elderberry (Sambucus nigra L.) Fruit Extract Alleviates Oxidative Stress, Insulin Resistance, and Inflammation in Hypertrophied 3T3-L1 Adipocytes and Activated RAW 264.7 Macrophages.

Oxidative stress and inflammation in hypertrophied adipose tissue with excessive fat accumulation play a crucial role in the development of obesity and accompanying metabolic dysfunctions. This study demonstrated the capacity of elderberry fruit (EDB) extract to decrease the elevated production of reactive oxygen species in hypertrophied 3T3-L1 adipocytes. Treatment with the EDB extract resulted in modulation of mRNA expression and protein secretion of key adipokines in hypertrophied adipocytes. Expression of leptin and adiponectin was, respectively, down- and up-regulated. Moreover, glucose uptake stimulation was noticed in mature adipocytes, both sensitive to insulin and insulin resistant. This may suggest a positive effect of EDB extract on insulin resistance status. The extract was also found to alleviate the inflammatory response in activated RAW 264.7 macrophages by down-regulating the expression of proinflammatory genes (TNF-α, IL-6, COX-2, iNOS) and suppressing the enhanced production of inflammatory mediators (TNF-α, IL-6, PGE2, NO). In vitro experiments showed that the EDB extract could inhibit digestive enzymes, including α-amylase, α-glucosidase, and pancreatic lipase, leading to reduced intestinal absorption of dietary lipids and carbohydrates. Further in vivo studies could be postulated to support EDB as a functional food component for the prevention and treatment of obesity and metabolic-immune comorbidities.