Evidence from preclinical and clinical metabolomics studies on the antidepressant effects of ketamine and esketamine.

The antidepressant effects of ketamine and esketamine are well-documented. Nonetheless, most of the underlying molecular mechanisms have to be uncovered yet. In the last decade, metabolomics has emerged as a useful means to investigate the metabolic phenotype associated with depression as well as changes induced by antidepressant treatments. This mini-review aims at summarizing the main findings from preclinical and clinical studies that used metabolomics to investigate the metabolic effects of subanesthetic, antidepressant doses of ketamine and esketamine and their relationship with clinical response. Both animal and human studies report alterations in several metabolic pathways - including the tricarboxylic acid cycle, glycolysis, the pentose phosphate pathway, lipid metabolism, amino acid metabolism, the kynurenine pathway, and the urea cycle - following the administration of ketamine or its enantiomers. Although more research is needed to clarify commonalities and differences in molecular mechanisms of action between the racemic compound and its enantiomers, these findings comprehensively support an influence of ketamine and esketamine on mitochondrial and cellular energy production, membrane homeostasis, neurotransmission, and signaling. Metabolomics may thus represent a promising strategy to clarify molecular mechanisms underlying treatment-resistant depression and related markers of clinical response to ketamine and esketamine. This body of preclinical and clinical evidence, if further substantiated, has the potential to guide clinicians towards personalized approaches, contributing to new paradigms in the clinical management of depression.

Revolutionizing Blood Collection: Innovations, Applications, and the Potential of Microsampling Technologies for Monitoring Metabolites and Lipids.

Blood serves as the primary global biological matrix for health surveillance, disease diagnosis, and response to drug treatment, holding significant promise for personalized medicine. The diverse array of lipids and metabolites in the blood provides a snapshot of both physiological and pathological processes, with many routinely monitored during conventional wellness checks. The conventional method involves intravenous blood collection, extracting a few milliliters via venipuncture, a technique limited to clinical settings due to its dependence on trained personnel. Microsampling methods have evolved to be less invasive (collecting ≤150 µL of capillary blood), user-friendly (enabling self-collection), and suitable for remote collection in longitudinal studies. Dried blood spot (DBS), a pioneering microsampling technique, dominates clinical and research domains. Recent advancements in device technology address critical limitations of classical DBS, specifically variations in hematocrit and volume. This review presents a comprehensive overview of state-of-the-art microsampling devices, emphasizing their applications and potential for monitoring metabolites and lipids in blood. The scope extends to diverse areas, encompassing population studies, nutritional investigations, drug discovery, sports medicine, and multi-omics research.

Conditioned Medium of Mesenchymal Stromal Cells Loaded with Paclitaxel Is Effective in Preclinical Models of Triple-Negative Breast Cancer (TNBC).

Triple-negative breast cancer (TNBC) is a very aggressive disease even in its early stages and is characterized by a severe prognosis. Neoadjuvant chemotherapy is one of the milestones of treatment, and paclitaxel (PTX) is among the most active drugs used in this setting. However, despite its efficacy, peripheral neuropathy occurs in approximately 20-25% of cases and represents the dose-limiting toxicity of this drug. New deliverable strategies to ameliorate drug delivery and reduce side effects are keenly awaited to improve patients' outcomes. Mesenchymal stromal cells (MSCs) have recently been demonstrated as promising drug delivery vectors for cancer treatment. The aim of the present preclinical study is to explore the possibility of a cell therapy approach based on the use of MSCs loaded with PTX to treat TNBC-affected patients. For this purpose, we in vitro evaluated the viability, migration and colony formation of two TNBC cell lines, namely, MDA-MB-231 and BT549, treated with MSC-PTX conditioned medium (MSC-CM PTX) in comparison with both CM of MSCs not loaded with PTX (CTRL) and free PTX. We observed stronger inhibitory effects on survival, migration and tumorigenicity for MSC-CM PTX than for CTRL and free PTX in TNBC cell lines. Further studies will provide more information about activity and potentially open the possibility of using this new drug delivery vector in the context of a clinical study.

VAMS-Based Blood Capillary Sampling for Mass Spectrometry-Based Human Metabolomics Studies.

Volumetric absorptive microsampling (VAMS) is a recently developed sample collection method that enables single-drop blood collection in a minimally invasive manner. Blood biomolecules can then be extracted and processed for analysis using several analytical platforms. The integration of VAMS with conventional mass spectrometry (MS)-based metabolomics approaches is an attractive solution for human studies representing a less-invasive procedure compared to phlebotomy with the additional potential for remote sample collection. However, as we recently demonstrated, VAMS samples require long-term storage at -80 °C. This study investigated the stability of VAMS samples during short-term storage and compared the metabolome obtained from capillary blood collected from the fingertip to those of plasma and venous blood from 22 healthy volunteers. Our results suggest that the blood metabolome collected by VAMS samples is stable at room temperature only for up to 6 h requiring subsequent storage at -80 °C to avoid significant changes in the metabolome. We also demonstrated that capillary blood provides better coverage of the metabolome compared to plasma enabling the analysis of several intracellular metabolites presented in red blood cells. Finally, this work demonstrates that with the appropriate pre-analytical protocol capillary blood can be successfully used for untargeted metabolomics studies.

Pharmacometabolomics for the Study of Lipid-Lowering Therapies: Opportunities and Challenges.

Lipid-lowering therapies are widely used to prevent the development of atherosclerotic cardiovascular disease (ASCVD) and related mortality worldwide. "Omics" technologies have been successfully applied in recent decades to investigate the mechanisms of action of these drugs, their pleiotropic effects, and their side effects, aiming to identify novel targets for future personalized medicine with an improvement of the efficacy and safety associated with the treatment. Pharmacometabolomics is a branch of metabolomics that is focused on the study of drug effects on metabolic pathways that are implicated in the variation of response to the treatment considering also the influences from a specific disease, environment, and concomitant pharmacological therapies. In this review, we summarized the most significant metabolomic studies on the effects of lipid-lowering therapies, including the most commonly used statins and fibrates to novel drugs or nutraceutical approaches. The integration of pharmacometabolomics data with the information obtained from the other "omics" approaches could help in the comprehension of the biological mechanisms underlying the use of lipid-lowering drugs in view of defining a precision medicine to improve the efficacy and reduce the side effects associated with the treatment.

Dual Functionalized Liposomes for Selective Delivery of Poorly Soluble Drugs to Inflamed Brain Regions.

Dual functionalized liposomes were developed to cross the blood−brain barrier (BBB) and to release their cargo in a pathological matrix metalloproteinase (MMP)-rich microenvironment. Liposomes were surface-functionalized with a modified peptide deriving from the receptor-binding domain of apolipoprotein E (mApoE), known to promote cargo delivery to the brain across the BBB in vitro and in vivo; and with an MMP-sensitive moiety for an MMP-triggered drug release. Different MMP-sensitive peptides were functionalized at both ends with hydrophobic stearate tails to yield MMP-sensitive lipopeptides (MSLPs), which were assembled into mApoE liposomes. The resulting bi-functional liposomes (i) displayed a < 180 nm diameter with a negative ζ-potential; (ii) were able to cross an in vitro BBB model with an endothelial permeability of 3 ± 1 × 10−5 cm/min; (iii) when exposed to functional MMP2 or 9, efficiently released an encapsulated fluorescein dye; (iv) showed high biocompatibility when tested in neuronal cultures; and (v) when loaded with glibenclamide, a drug candidate with poor aqueous solubility, reduced the release of proinflammatory cytokines from activated microglial cells.

Spatial Multiomics of Lipids, N-Glycans, and Tryptic Peptides on a Single FFPE Tissue Section.

Mass spectrometry imaging (MSI) is an emerging technology that is capable of mapping various biomolecules within their native spatial context, and performing spatial multiomics on formalin-fixed paraffin-embedded (FFPE) tissues may further increase the molecular characterization of pathological states. Here we present a novel workflow which enables the sequential MSI of lipids, N-glycans, and tryptic peptides on a single FFPE tissue section and highlight the enhanced molecular characterization that is offered by combining the multiple spatial omics data sets. In murine brain and clear cell renal cell carcinoma (ccRCC) tissue, the three molecular levels provided complementary information and characterized different histological regions. Moreover, when the spatial omics data was integrated, the different histopathological regions of the ccRCC tissue could be better discriminated with respect to the imaging data set of any single omics class. Taken together, these promising findings demonstrate the capability to more comprehensively map the molecular complexity within pathological tissue.

Identification of mechanically separated meat using multivariate analysis of 43 trace elements detected by inductively coupled mass spectrometry: A validated approach.

The European Food Safety Authority asked for novel approaches for identifying mechanically separated meat (MSM) in meat products, due to food safety concern. In this study, a novel approach based on multivariate analysis of 43 trace elements in meat products is described. Overall, 27 trace elements and 16 rare earth elements were determined by using ICP-MS after sample mineralization of 100 meat samples, composed of different percentages of MSM, obtained at low and high pressure, and without MSM. After development and optimization, the multivariate approach was validated by analyzing and then classifying 10 "blind" meat samples, obtaining method accuracy equal to 90%. Thus, the applicability of this new analytical approach was demonstrated. The method represents a significant improvement for this type of determination, especially when MSM is obtained at low pressure, since this product is characterized by chemical characteristics very similar to fresh meat.

High Intensity Concentric-Eccentric Exercise Under Hypoxia Changes the Blood Metabolome of Trained Athletes.

The aim of this study was to determine alterations of the metabolome in blood plasma in response to concentric-eccentric leg exercise performed at a simulated altitude of 3,500 m. To do so, we recruited 11 well-trained subjects and performed an untargeted metabolomics analysis of plasma samples obtained before, 20 min after as well as on day 8 after five sets of maximal, concentric-eccentric leg exercises that lasted 90 s each. We identified and annotated 115 metabolites through untargeted liquid chromatography-mass spectrometry metabolomics and used them to further calculate 20 sum/ratio of metabolites. A principal component analysis (PCA) revealed differences in-between the overall metabolome at rest and immediately after exercise. Interestingly, some systematic changes of relative metabolite concentrations still persisted on day 8 after exercise. The first two components of the PCA explained 34% of the relative concentrations of all identified metabolites analyzed together. A volcano plot indicates that 35 metabolites and two metabolite ratios were significantly changed directly after exercise, such as metabolites related to carbohydrate and TCA metabolism. Moreover, we observed alterations in the relative concentrations of amino acids (e.g., decreases of valine, leucine and increases in alanine) and purines (e.g., increases in hypoxanthine, xanthine and uric acid). In summary, high intensity concentric-eccentric exercise performed at simulated altitude systematically changed the blood metabolome in trained athletes directly after exercise and some relative metabolite concentrations were still changed on day 8. The importance of that persisting metabolic alterations on exercise performance should be studied further.

Whole Exome Sequencing Enhanced Imputation Identifies 85 Metabolite Associations in the Alpine CHRIS Cohort.

Metabolites are intermediates or end products of biochemical processes involved in both health and disease. Here, we take advantage of the well-characterized Cooperative Health Research in South Tyrol (CHRIS) study to perform an exome-wide association study (ExWAS) on absolute concentrations of 175 metabolites in 3294 individuals. To increase power, we imputed the identified variants into an additional 2211 genotyped individuals of CHRIS. In the resulting dataset of 5505 individuals, we identified 85 single-variant genetic associations, of which 39 have not been reported previously. Fifteen associations emerged at ten variants with >5-fold enrichment in CHRIS compared to non-Finnish Europeans reported in the gnomAD database. For example, the CHRIS-enriched ETFDH stop gain variant p.Trp286Ter (rs1235904433-hexanoylcarnitine) and the MCCC2 stop lost variant p.Ter564GlnextTer3 (rs751970792-carnitine) have been found in patients with glutaric acidemia type II and 3-methylcrotonylglycinuria, respectively, but the loci have not been associated with the respective metabolites in a genome-wide association study (GWAS) previously. We further identified three gene-trait associations, where multiple rare variants contribute to the signal. These results not only provide further evidence for previously described associations, but also describe novel genes and mechanisms for diseases and disease-related traits.

GCN5 contributes to intracellular lipid accumulation in human primary cardiac stromal cells from patients affected by Arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.

Therapeutic induction of energy metabolism reduces neural tissue damage and increases microglia activation in severe spinal cord injury.

Neural tissue has high metabolic requirements. Following spinal cord injury (SCI), the damaged tissue suffers from a severe metabolic impairment, which aggravates axonal degeneration and neuronal loss. Impaired cellular energetic, tricarboxylic acid (TCA) cycle and oxidative phosphorylation metabolism in neuronal cells has been demonstrated to be a major cause of neural tissue death and regeneration failure following SCI. Therefore, rewiring the spinal cord cell metabolism may be an innovative therapeutic strategy for the treatment of SCI. In this study, we evaluated the therapeutic effect of the recovery of oxidative metabolism in a mouse model of severe contusive SCI. Oral administration of TCA cycle intermediates, co-factors, essential amino acids, and branched-chain amino acids was started 3 days post-injury and continued until the end of the experimental procedures. Metabolomic, immunohistological, and biochemical analyses were performed on the injured spinal cord sections. Administration of metabolic precursors enhanced spinal cord oxidative metabolism. In line with this metabolic shift, we observed the activation of the mTORC1 anabolic pathway, the increase in mitochondrial mass, and ROS defense which effectively prevented the injury-induced neural cell apoptosis in treated animals. Consistently, we found more choline acetyltransferase (ChAT)-expressing motor neurons and increased neurofilament-positive corticospinal axons in the spinal cord parenchyma of the treated mice. Interestingly, oral administration of the metabolic precursors increased the number of activated microglia expressing the CD206 marker suggestive of a pro-resolutive, M2-like phenotype. These molecular and histological modifications observed in treated animals ultimately led to a significant, although partial, improvement of the motor functions. Our data demonstrate that rewiring the cellular metabolism can represent an effective strategy to treat SCI.

Ion mobility mass spectrometry in the omics era: Challenges and opportunities for metabolomics and lipidomics.

Researchers worldwide are taking advantage of novel, commercially available, technologies, such as ion mobility mass spectrometry (IM-MS), for metabolomics and lipidomics applications in a variety of fields including life, biomedical, and food sciences. IM-MS provides three main technical advantages over traditional LC-MS workflows. Firstly, in addition to mass, IM-MS allows collision cross-section values to be measured for metabolites and lipids, a physicochemical identifier related to the chemical shape of an analyte that increases the confidence of identification. Second, IM-MS increases peak capacity and the signal-to-noise, improving fingerprinting as well as quantification, and better defining the spatial localization of metabolites and lipids in biological and food samples. Third, IM-MS can be coupled with various fragmentation modes, adding new tools to improve structural characterization and molecular annotation. Here, we review the state-of-the-art in IM-MS technologies and approaches utilized to support metabolomics and lipidomics applications and we assess the challenges and opportunities in this growing field.

A High-Throughput HILIC-MS-Based Metabolomic Assay for the Analysis of Polar Metabolites.

Mass spectrometry (MS)-based metabolomics approaches have been used for characterizing the metabolite content and composition of biological samples in drug discovery and development, as well as in metabolic engineering, and food and plant sciences applications. Here, we describe a protocol routinely used in our laboratory to conduct a metabolic profiling of small polar metabolites from biological samples. Metabolites can be extracted from each sample using a methanol-based single-phase extraction procedure. The combination of LC-based hydrophilic interaction liquid chromatography (HILIC) and a hybrid quadrupole-time of flight (Q-ToF) mass spectrometer allows the comprehensive analysis of small polar metabolites including sugars, phosphorylated compounds, purines and pyrimidines, nucleotides, nucleosides, acylcarnitines, carboxylic acids, hydrophilic vitamins and amino acids. Retention times and accurate masses of metabolites involved in key metabolic pathways are annotated for routine high-throughput screening in both untargeted and targeted metabolomics analyses.

Lipidomic Typing of Colorectal Cancer Tissue Containing Tumour-Infiltrating Lymphocytes by MALDI Mass Spectrometry Imaging.

Predicting the prognosis of colorectal cancer (CRC) patients remains challenging and a characterisation of the tumour immune environment represents one of the most crucial avenues when attempting to do so. For this reason, molecular approaches which are capable of classifying the immune environments associated with tumour infiltrating lymphocytes (TILs) are being readily investigated. In this proof of concept study, we aim to explore the feasibility of using spatial lipidomics by MALDI-MSI to distinguish CRC tissue based upon their TIL content. Formalin-fixed paraffin-embedded tissue from human thymus and tonsil was first analysed by MALDI-MSI to obtain a curated mass list from a pool of single positive T lymphocytes, whose putative identities were annotated using an LC-MS-based lipidomic approach. A CRC tissue microarray (TMA, n = 30) was then investigated to determine whether these cases could be distinguished based upon their TIL content in the tumour and its microenvironment. MALDI-MSI from the pool of mature T lymphocytes resulted in the generation of a curated mass list containing 18 annotated m/z features. Initially, subsets of T lymphocytes were then distinguished based on their state of maturation and differentiation in the human thymus and tonsil tissue. Then, when applied to a CRC TMA containing differing amounts of T lymphocyte infiltration, those cases with a high TIL content were distinguishable from those with a lower TIL content, especially within the tumour microenvironment, with three lipid signals being shown to have the greatest impact on this separation (p < 0.05). On the whole, this preliminary study represents a promising starting point and suggests that a lipidomics MALDI-MSI approach could be a promising tool for subtyping the diverse immune environments in CRC.

Metabolic Signature of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is a genetic-based cardiac disease accompanied by severe ventricular arrhythmias and a progressive substitution of the myocardium with fibro-fatty tissue. ACM is often associated with sudden cardiac death. Due to the reduced penetrance and variable expressivity, the presence of a genetic defect is not conclusive, thus complicating the diagnosis of ACM. Recent studies on human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) obtained from ACM individuals showed a dysregulated metabolic status, leading to the hypothesis that ACM pathology is characterized by an impairment in the energy metabolism. However, despite efforts having been made for the identification of ACM specific biomarkers, there is still a substantial lack of information regarding the whole metabolomic profile of ACM patients. The aim of the present study was to investigate the metabolic profiles of ACM patients compared to healthy controls (CTRLs). The targeted Biocrates AbsoluteIDQ® p180 assay was used on plasma samples. Our analysis showed that ACM patients have a different metabolome compared to CTRLs, and that the pathways mainly affected include tryptophan metabolism, arginine and proline metabolism and beta oxidation of fatty acids. Altogether, our data indicated that the plasma metabolomes of arrhythmogenic cardiomyopathy patients show signs of endothelium damage and impaired nitric oxide (NO), fat, and energy metabolism.

Antigen Retrieval and Its Effect on the MALDI-MSI of Lipids in Formalin-Fixed Paraffin-Embedded Tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue represents the primary source of clinical tissue and is routinely used in MALDI-MSI studies. However, it is not particularly suitable for lipidomics imaging given that many species are depleted during tissue processing. Irrespective, a number of solvent-resistant lipids remain, but their extraction may be hindered by the cross-link between proteins. Therefore, an antigen retrieval step could enable the extraction of a greater number of lipids and may provide information that is complementary to that which can be obtained from other biomolecules, such as proteins. In this short communication, we aim to address the effect of performing antigen retrieval prior to MALDI-MSI of lipids in FFPE tissue. As a result, an increased number of lipid signals could be detected and may have derived from lipid species that are known to be implicated in the lipid-protein cross-linking that is formed as a result of formalin fixation. Human renal cancer tissue was used as a proof of concept to determine whether using these detected lipid signals were also able to highlight the histopathological regions that were present. These preliminary findings may highlight the potential to enhance the clinical relevance of the lipidomic information obtained from FFPE tissue.

Metabolic reprogramming of Salmonella infected macrophages and its modulation by iron availability and the mTOR pathway.

Iron is an essential nutrient for immune cells and microbes, therefore the control of its homeostasis plays a decisive role for infections. Moreover, iron affects metabolic pathways by modulating the translational expression of the key tricarboxylic acid cycle (TCA) enzyme mitochondrial aconitase and the energy formation by mitochondria. Recent data provide evidence for metabolic re-programming of immune cells including macrophages during infection which is centrally controlled by mTOR. We herein studied the effects of iron perturbations on metabolic profiles in macrophages upon infection with the intracellular bacterium Salmonella enterica serovar Typhimurium and analysed for a link to the mTOR pathway. Infection of the murine macrophage cell line RAW264.7 with Salmonella resulted in the induction of mTOR activity, anaerobic glycolysis and inhibition of the TCA activity as reflected by reduced pyruvate and increased lactate levels. In contrast, iron supplementation to macrophages not only affected the mRNA expression of TCA and glycolytic enzymes but also resulted in metabolic reprogramming with increased pyruvate accumulation and reduced lactate levels apart from modulating the concentrations of several other metabolites. While mTOR slightly affected cellular iron homeostasis in infected macrophages, mTOR inhibition by rapamycin resulted in a significant growth promotion of bacteria. Importantly, iron further increased bacterial numbers in rapamycin treated macrophages, however, the metabolic profiles induced by iron in the presence or absence of mTOR activity differed in several aspects. Our data indicate, that iron not only serves as a bacterial nutrient but also acts as a metabolic modulator of the TCA cycle, partly reversing the Warburg effect and resulting in a pathogen friendly nutritional environment.

Lipidomics, Atrial Conduction, and Body Mass Index.

BACKGROUND: Lipids are increasingly involved in cardiovascular risk prediction as potential proarrhythmic influencers. However, knowledge is limited about the specific mechanisms connecting lipid alterations with atrial conduction. METHODS: To shed light on this issue, we conducted a broad assessment of 151 sphingo- and phospholipids, measured using mass spectrometry, for association with atrial conduction, measured by P wave duration (PWD) from standard electrocardiograms, in the MICROS study (Microisolates in South Tyrol) (n=839). Causal pathways involving lipidomics, body mass index (BMI), and PWD were assessed using 2-sample Mendelian randomization analyses based on published genome-wide association studies of lipidomics (n=4034) and BMI (n=734 481), and genetic association analysis of PWD in 5 population-based studies (n=24 236). RESULTS: We identified an association with relative phosphatidylcholine 38:3 (%PC 38:3) concentration, which was replicated in the ORCADES (Orkney Complex Disease Study; n=951), with a pooled association across studies of 2.59 (95% CI, 1.3-3.9; P=1.1×10-4) ms PWD per mol% increase. While being independent of cholesterol, triglycerides, and glucose levels, the %PC 38:3-PWD association was mediated by BMI. Results supported a causal effect of BMI on both PWD ( P=8.3×10-5) and %PC 38:3 ( P=0.014). CONCLUSIONS: Increased %PC 38:3 levels are consistently associated with longer PWD, partly because of the confounding effect of BMI. The causal effect of BMI on PWD reinforces evidence of BMI's involvement into atrial electrical activity.