CE-MS-Based Clinical Metabolomics of Human Plasma.

Capillary electrophoresis coupled to mass spectrometry (CE-MS) has emerged as a powerful analytical technique with significant implications for clinical research and diagnostics. The integration of information from CE and MS strengthens confidence in the identification of compounds present in clinical samples. The ability of CE to separate molecules based on their electrophoretic mobility coupled to MS enables the accurate identification and quantification of analytes, even in complex biological matrices such as human plasma.Here, we present a detailed protocol for an untargeted metabolomics study using CE-MS and its application in a study on human plasma from patients suffering Long COVID syndrome. The protocol ranges from sample preparation to biological interpretation, detailing a workflow enabling the analysis of cationic and anionic compounds, metabolite identification, and data processing.

New insight into primary hyperparathyroidism using untargeted metabolomics.

Primary Hyperparathyroidism (PHPT) is characterized by excessive parathormone (PTH) secretion and disrupted calcium homeostasis. Untargeted metabolomics offers a valuable approach to understanding the complex metabolic alterations associated with different diseases, including PHPT. Plasma untargeted metabolomics was applied to investigate the metabolic profiles of PHPT patients compared to a control group. Two complementary liquid-phase separation techniques were employed to comprehensively explore the metabolic landscape in this retrospective, single-center study. The study comprised 28 female patients diagnosed following the current guidelines of PHPT diagnosis and a group of 30 healthy females as a control group. To evaluate their association with PHPT, we identified changes in plasma metabolic profiles in patients with PHPT compared to the control group. The primary outcome measure included detecting plasma metabolites and discriminating PHPT patients from controls. The study unveiled specific metabolic imbalances that may link L-amino acids with peptic ulcer disease, gamma-glutamyls with oxidative stress, and asymmetric dimethylarginine (ADMA) with cardiovascular complications. Several metabolites, such as gamma-glutamyls, caffeine, sex hormones, carnitine, sphingosine-1-phosphate (S-1-P), and steroids, were connected with reduced bone mineral density (BMD). Metabolic profiling identified distinct metabolic patterns between patients with PHPT and healthy controls. These findings provided valuable insights into the pathophysiology of PHPT.

Probiotic Lactobacillus plantarum 299v supplementation in patients with major depression in a double-blind, randomized, placebo-controlled trial: A metabolomics study.

BACKGROUND: Understanding the multifactorial nature of major depressive disorder (MDD) is crucial for tailoring treatments. However, the complex interplay of various factors underlying the development and progression of MDD poses significant challenges. Our previous study demonstrated improvements in cognitive functions in MDD patients undergoing treatment with selective serotonin reuptake inhibitors (SSRIs) supplemented with Lactobacillus plantarum 299v (LP299v). METHODS: To elucidate the biochemical mechanisms underlying cognitive functions improvements, we explored underlying metabolic changes. We employed multi-platform metabolomics, including LC-QTOF-MS and CE-TOF-MS profiling, alongside chiral LC-QqQ-MS analysis for amino acids. RESULTS: Supplementation of SSRI treatment with LP299v intensified the reduction of long-chain acylcarnitines, potentially indicating improved mitochondrial function. LP299v supplementation reduced N-acyl taurines more than four times compared to the placebo, suggesting a substantial impact on restoring biochemical balance. The LP299v-supplemented group showed increased levels of oxidized glycerophosphocholine (oxPC). Additionally, LP299v supplementation led to higher levels of sphingomyelins, L-histidine, D-valine, and p-cresol. LIMITATIONS: This exploratory study suggests potential metabolic pathways influenced by LP299v supplementation. However, the need for further research hinders the ability to draw definitive conclusions. CONCLUSIONS: Observed metabolic changes were linked to mitochondrial dysfunction, inflammation, oxidative stress, and gut microbiota disruption. Despite the subtle nature of this alterations, our research successfully detected these differences and connected them to the metabolic disruptions associated with MDD. Our findings emphasise the intricate relationship between metabolism, gut microbiota, and mental health prompting further research into the mechanisms of action of probiotics in MDD treatment.

Untargeted metabolomics analysis of serum and urine unveils the protective effect of cilastatin on altered metabolic pathways during cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is one of the most serious complications of cisplatin anticancer therapies. Cilastatin is a highly promising nephroprotective agent to eventually enter clinical use, but its biochemical mechanism is still not fully understood. We have employed an untargeted metabolomics approach based on capillary electrophoresis mass spectrometry (CE-MS) analysis of serum and urine from an in vivo rat model, to explore the metabolic pathways involved in cisplatin-induced AKI and cilastatin nephroprotection. A total of 155 and 76 identified metabolites were found to be significantly altered during cisplatin treatment in urine and serum, respectively. Most of these altered metabolites were either partially or totally recovered by cilastatin and cisplatin co-treatment. The main metabolic pathways disturbed by cisplatin during AKI involved diverse amino acids metabolism and biosynthesis, tricarboxylic acids (TCA) cycle, nicotinate and nicotinamide metabolism, among others. Cilastatin was proved to protect diverse cisplatin-altered pathways involving metabolites related to immunomodulation, inflammation, oxidative stress and amino acid metabolism in proximal tubules. However, cisplatin-altered mitochondrial metabolism (especially, the energy-producing TCA cycle) remained largely unprotected by cilastatin, suggesting an unresolved mitochondrial direct damage. Multivariate analysis allowed effective discrimination of cisplatin-induced AKI and cilastatin renoprotection based on metabolic features. A number of potential serum and urine biomarkers could also be foreseen for cisplatin-induced AKI detection and cilastatin nephroprotection.

Metabolomics for searching validated biomarkers in cancer studies: a decade in review.

INTRODUCTION: In the dynamic landscape of modern healthcare, the ability to anticipate and diagnose diseases, particularly in cases where early treatment significantly impacts outcomes, is paramount. Cancer, a complex and heterogeneous disease, underscores the critical importance of early diagnosis for patient survival. The integration of metabolomics information has emerged as a crucial tool, complementing the genotype-phenotype landscape and providing insights into active metabolic mechanisms and disease-induced dysregulated pathways. AREAS COVERED: This review explores a decade of developments in the search for biomarkers validated within the realm of cancer studies. By critically assessing a diverse array of research articles, clinical trials, and studies, this review aims to present an overview of the methodologies employed and the progress achieved in identifying and validating biomarkers in metabolomics results for various cancer types. EXPERT OPINION: Through an exploration of more than 800 studies, this review has allowed to establish a general idea about state-of-art in the search of biomarkers in metabolomics studies involving cancer which include certain level of results validation. The potential for metabolites as diagnostic markers to reach the clinic and make a real difference in patient health is substantial, but challenges remain to be explored.

Application of a capillary electrophoresis-mass spectrometry metabolomics workflow in zebrafish larvae reveals new effects of cortisol.

In contemporary biomedical research, the zebrafish (Danio rerio) is increasingly considered a model system, as zebrafish embryos and larvae can (potentially) fill the gap between cultured cells and mammalian animal models, because they can be obtained in large numbers, are small and can easily be manipulated genetically. Given that capillary electrophoresis-mass spectrometry (CE-MS) is a useful analytical separation technique for the analysis of polar ionogenic metabolites in biomass-limited samples, the aim of this study was to develop and assess a CE-MS-based analytical workflow for the profiling of (endogenous) metabolites in extracts from individual zebrafish larvae and pools of small numbers of larvae. The developed CE-MS workflow was used to profile metabolites in extracts from pools of 1, 2, 4, 8, 12, 16, 20, and 40 zebrafish larvae. For six selected endogenous metabolites, a linear response (R2  > 0.98) for peak areas was obtained in extracts from these pools. The repeatability was satisfactory, with inter-day relative standard deviation values for peak area of 9.4%-17.7% for biological replicates (n = 3 over 3 days). Furthermore, the method allowed the analysis of over 70 endogenous metabolites in a pool of 12 zebrafish larvae, and 29 endogenous metabolites in an extract from only 1 zebrafish larva. Finally, we applied the optimized CE-MS workflow to identify potential novel targets of the mineralocorticoid receptor in mediating the effects of cortisol.

Metabolomic profile of neuroendocrine tumors identifies methionine, porphyrin, and tryptophan metabolisms as key dysregulated pathways associated with patient survival.

OBJECTIVE: Metabolic profiling is a valuable tool to characterize tumor biology but remains largely unexplored in neuroendocrine tumors (NETs). Our aim was to comprehensively assess the metabolomic profile of NETs and identify novel prognostic biomarkers and dysregulated molecular pathways. DESIGN AND METHODS: Multiplatform untargeted metabolomic profiling (GC-MS, CE-MS, and LC-MS) was performed in plasma from 77 patients with G1-2 extra-pancreatic NETs enrolled in the AXINET trial (NCT01744249) (study cohort) and from 68 non-cancer individuals (control). The prognostic value of each differential metabolite (n = 155) in NET patients (P < .05) was analyzed by univariate and multivariate analyses adjusted for multiple testing and other confounding factors. Related pathways were explored by Metabolite Set Enrichment Analysis (MSEA) and Metabolite Pathway Analysis (MPA). RESULTS: Thirty-four metabolites were significantly associated with progression-free survival (PFS) (n = 16) and/or overall survival (OS) (n = 27). Thirteen metabolites remained significant independent prognostic factors in multivariate analysis, 3 of them with a significant impact on both PFS and OS. Unsupervised clustering of these 3 metabolites stratified patients in 3 distinct prognostic groups (1-year PFS of 71.1%, 47.7%, and 15.4% (P = .012); 5-year OS of 69.7%, 32.5%, and 27.7% (P = .003), respectively). The MSEA and MPA of the 13-metablolite signature identified methionine, porphyrin, and tryptophan metabolisms as the 3 most relevant dysregulated pathways associated with the prognosis of NETs. CONCLUSIONS: We identified a metabolomic signature that improves prognostic stratification of NET patients beyond classical prognostic factors for clinical decisions. The enriched metabolic pathways identified reveal novel tumor vulnerabilities that may foster the development of new therapeutic strategies for these patients.

A host-specific diaminobutyrate aminotransferase contributes to symbiotic performance, homoserine metabolism, and competitiveness in the Rhizobium leguminosarum/Pisum sativum system.

Rhizobium leguminosarum bv. viciae (Rlv) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a diaminobutyrate-2-oxoglutarate aminotransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor ß-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere.

Mapping early serum proteome signatures of liver regeneration in living donor liver transplant cases.

The liver is the only solid organ capable of regenerating itself to regain 100% of its mass and function after liver injury and/or partial hepatectomy (PH). This exceptional property represents a therapeutic opportunity for severe liver disease patients. However, liver regeneration (LR) might fail due to poorly understood causes. Here, we have investigated the regulation of liver proteome and phosphoproteome at a short time after PH (9 h), to depict a detailed mechanistic background of the early LR phase. Furthermore, we analyzed the dynamic changes of the serum proteome and metabolome of healthy living donor liver transplant (LDLT) donors at different time points after surgery. The molecular profiles from both analyses were then correlated. Insulin and FXR-FGF15/19 signaling were stimulated in mouse liver after PH, leading to the activation of the main intermediary kinases (AKT and ERK). Besides, inhibition of the hippo pathway led to an increased expression of its target genes and of one of its intermediary proteins (14-3-3 protein), contributing to cell proliferation. In association with these processes, metabolic reprogramming coupled to enhanced mitochondrial activity cope for the energy and biosynthetic requirements of LR. In human serum of LDLT donors, we identified 56 proteins and 13 metabolites statistically differential which recapitulate some of the main cellular processes orchestrating LR in its early phase. These results provide mechanisms and protein mediators of LR that might prove useful for the follow-up of the regenerative process in the liver after PH as well as preventing the occurrence of complications associated with liver resection.

Optimization of capillary electrophoresis coupled to negative mode electrospray ionization-mass spectrometry using polyvinyl alcohol coated capillaries. Application to a study on non-small cell lung cancer.

Despite recent developments in separation techniques, the analysis of relatively small highly polar negatively charged analytes (e.g. small organic acids, phosphorylated sugars, and underivatized amino acids) remains challenging. Capillary electrophoresis coupled to mass spectrometry (CE-MS) has been included in the untargeted metabolomics toolbox, although mostly in positive polarity. The aim of this study was to assess the use of CE-MS to analyze highly polar and negatively charged metabolites at physiological levels without the need for derivatization. After a preliminary selection, conditions regarding CE (buffers, applied potential, injection time and applied pressure), electrospray parameters (sheath liquid flow, temperature and drying gas flow, nebulizer, and capillary voltage), and fragmentor voltage were optimized using a capillary coated with polyvinyl alcohol (PVA) for the metabolic profiling of anionic compounds compared to fused silica as the reference capillary. In addition, a database of 240 metabolites with two relative migration times (RMT) obtained against methionine sulfone and 2-morpholinoethanesulfonic acid (MES) as internal standards (IS) has been compiled. Finally, the optimized method has been used to characterize the metabolic profile of blood plasma in patients with non-small cell lung cancer (NSCLC). The identified compounds are mostly amino acids and their derivatives, carboxylic acids and organic compounds from the TCA cycle, and sugars and their phosphoderivates. In addition, we performed a comparative study to find significant differences between non-small cell lung cancer (NSCLC) vs non-cancer individuals, and squamous cell carcinoma (SCC) and adenocarcinoma (ADC) vs non-cancer individuals, respectively, searching for differences between the various types of NSCLC.