Metabolomic profile of acute myeloid leukaemia parallels of prognosis and response to therapy.

The heterogeneity of acute myeloid leukemia (AML), a complex hematological malignancy, is caused by mutations in myeloid cells affecting their differentiation and proliferation. Thus, various cytogenetic alterations in AML cells may be characterized by a unique metabolome and require different treatment approaches. In this study, we performed untargeted metabolomics to assess metabolomics differences between AML patients and healthy controls, AML patients with different treatment outcomes, AML patients in different risk groups based on the 2017 European LeukemiaNet (ELN) recommendations for the diagnosis and management of AML, AML patients with and without FLT3-ITD mutation, and a comparison between patients with FLT3-ITD, CBF-AML (Core binding factor acute myelogenous leukemia), and MLL AML (mixed-lineage leukemia gene) in comparison to control subjects. Analyses were performed in serum samples using liquid chromatography coupled with mass spectrometry (LC-MS). The obtained metabolomics profiles exhibited many alterations in glycerophospholipid and sphingolipid metabolism and allowed us to propose biomarkers based on each of the above assessments as an aid for diagnosis and eventual classification, allowing physicians to choose the best-suited treatment approach. These results highlight the application of LC-MS-based metabolomics of serum samples as an aid in diagnostics and a potential minimally invasive prognostic tool for identifying various cytogenetic and treatment outcomes of AML.

Simultaneous Comparison of Aqueous Humor and Serum Metabolic Profiles of Diabetic and Nondiabetic Patients Undergoing Cataract Surgery-A Targeted and Quantitative Metabolomics Study.

The aim of this study was to compare the aqueous humor (AH) and serum concentrations of metabolites in diabetic (n = 36) and nondiabetic (n = 36) senior adults undergoing cataract surgery. Blood samples were collected before surgery and AH during surgery. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted metabolomic and lipidomic analyses of samples were performed using the AbsoluteIDQ® p180 kit. Out of 188 metabolites targeted by the kit, 41 and 133 were detected in >80% of AH and serum samples, respectively. Statistical analysis performed to indicate metabolites differentiating diabetic and nondiabetic patients showed 8 and 20 significant metabolites in AH and serum, respectively. Pathway analysis performed for significant metabolites revealed that galactose metabolism is mostly affected in the AH, while arginine biosynthesis is mostly affected in the serum. Among metabolites that differentiate diabetic and nondiabetic patients, arginine was the only metabolite common to both serum and AH samples, as well as the only one with a decreased concentration in both body fluids of diabetic patients. Concentrations of the rest were elevated in AH and lowered in serum. This may suggest different mechanisms of diabetes-related dysregulation of the local metabolism in the eye in comparison to systemic changes observed in the blood.

Extent of interocular (a)symmetry based on the metabolomic profile of human aqueous humor.

Aims: Interocular comparison of the metabolomic signature of aqueous humor (AH) was performed. The aim of the study was to quantitatively evaluate the symmetry in concentrations of various metabolites belonging to different categories. Methods: The study included AH samples from 23 patients, 74.17 ± 11.52 years old, undergoing simultaneous bilateral cataract surgery at the Ophthalmology Department of the Medical University of Bialystok, Poland. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics and lipidomics analyses of AH samples were performed using the AbsoluteIDQ® p180 kit. Out of 188 metabolites available in the kit, 67 were measured in the majority (>70%) of the samples: 21/21 amino acids, 10/22 biogenic amines, 9/40 acylcarnitines, 0/14 lysophosphatidylcholines, 21/76 phosphatidylcholines, 5/15 sphingolipids, and 1/1sum of hexoses. Results: The comparison of both eyes revealed that the concentrations of metabolites did not differ significantly (p < 0.05) except for taurine (p = 0.037). There was moderate-to-strong positive interocular correlation (r > 0.5) between most metabolites regarding concentration. This was confirmed by the high intraclass correlation coefficient (ICC) values of different levels, which varied for the different metabolites. However, there were exceptions. Correlations were not significant for 2 acylcarnitines (tiglylcarnitine and decadienylcarnitine) and 3 glycerophospholipids (PC aa C32:3, PC aa C40:2, and PC aa C40:5). Conclusion: With a few exceptions, a single eye was found to be representative of the fellow eye in terms of the concentration of most of the analyzed metabolites. The degree of intraindividual variability in the AH of fellow eyes differs for particular metabolites/metabolite categories.

Exploration of oxidized phosphocholine profile in non-small-cell lung cancer.

Introduction: Lung cancer is one of the most frequently studied types of cancer and represents the most common and lethal neoplasm. Our previous research on non-small cell lung cancer (NSCLC) has revealed deep lipid profile reprogramming and redox status disruption in cancer patients. Lung cell membranes are rich in phospholipids that are susceptible to oxidation, leading to the formation of bioactive oxidized phosphatidylcholines (oxPCs). Persistent and elevated levels of oxPCs have been shown to induce chronic inflammation, leading to detrimental effects. However, recent reports suggest that certain oxPCs possess anti-inflammatory, pro-survival, and endothelial barrier-protective properties. Thus, we aimed to measure the levels of oxPCs in NSCLC patients and investigate their potential role in lung cancer. Methods: To explore the oxPCs profiles in lung cancer, we performed in-depth, multi-level metabolomic analyses of nearly 350 plasma and lung tissue samples from 200 patients with NSCLC, including adenocarcinoma (ADC) and squamous cell carcinoma (SCC), the two most prevalent NSCLC subtypes and COPD patients as a control group. First, we performed oxPC profiling of plasma samples. Second, we analyzed tumor and non-cancerous lung tissues collected during the surgical removal of NSCLC tumors. Because of tumor tissue heterogeneity, subsequent analyses covered the surrounding healthy tissue and peripheral and central tumors. To assess whether the observed phenotypic changes in the patients were associated with measured oxPC levels, metabolomics data were augmented with data from medical records. Results: We observed a predominance of long-chain oxPCs in plasma samples and of short-chain oxPCs in tissue samples from patients with NSCLC. The highest concentration of oxPCs was observed in the central tumor region. ADC patients showed higher levels of oxPCs compared to the control group, than patients with SCC. Conclusion: The detrimental effects associated with the accumulation of short-chain oxPCs suggest that these molecules may have greater therapeutic utility than diagnostic value, especially given that elevated oxPC levels are a hallmark of multiple types of cancer.

Elevated level of lysophosphatidic acid among patients with HNF1B mutations and its role in RCAD syndrome: a multiomic study.

INTRODUCTION: Patients with hepatocyte nuclear factor-1 beta (HNF1B) mutations present a variable phenotype with two main symptoms: maturity onset diabetes of the young (MODY) and polycystic kidney disease (PKD). OBJECTIVES: Identification of serum metabolites specific for HNF1Bmut and evaluation of their role in disease pathogenesis. METHODS: We recruited patients with HNF1Bmut (N = 10), HNF1Amut (N = 10), PKD: non-dialyzed and dialyzed (N = 8 and N = 13); and healthy controls (N = 12). Serum fingerprinting was performed by LC-QTOF-MS. Selected metabolite was validated by ELISA (enzyme-linked immunosorbent assay) measurements and then biologically connected with HNF1B by in silico analysis. HepG2 were stimulated with lysophosphatidic acid (LPA) and HNF1B gene was knocked down (kd) by small interfering RNA. Transcriptomic analysis with microarrays and western blot measurements were performed. RESULTS: Serum levels of six metabolites including: arachidonic acid, hydroxyeicosatetraenoic acid, linoleamide and three LPA (18:1, 18:2 and 20:4), had AUC (the area under the curve) > 0.9 (HNF1Bmut vs comparative groups). The increased level of LPA was confirmed by ELISA measurements. In HepG2HNF1Bkd cells LPA stimulation lead to downregulation of many pathways associated with cell cycle, lipid metabolism, and upregulation of steroid hormone metabolism and Wnt signaling. Also, increased intracellular protein level of autotaxin was detected in the cells. GSK-3alpha/beta protein level and its phosphorylated ratio were differentially affected by LPA stimulation in HNF1Bkd and control cells. CONCLUSIONS: LPA is elevated in sera of patients with HNF1Bmut. LPA contributes to the pathogenesis of HNF1B-MODY by affecting Wnt/GSK-3 signaling.

The Ability of Metabolomics to Discriminate Non-Small-Cell Lung Cancer Subtypes Depends on the Stage of the Disease and the Type of Material Studied.

Identification of the NSCLC subtype at an early stage is still quite sophisticated. Metabolomics analysis of tissue and plasma of NSCLC patients may indicate new, and yet unknown, metabolic pathways active in the NSCLC. Our research characterized the metabolomics profile of tissue and plasma of patients with early and advanced NSCLC stage. Samples were subjected to thorough metabolomics analyses using liquid chromatography-mass spectrometry (LC-MS) technique. Tissue and/or plasma samples from 137 NSCLC patients were analyzed. Based on the early stage tissue analysis, more than 200 metabolites differentiating adenocarcinoma (ADC) and squamous cell lung carcinoma (SCC) subtypes as well as normal tissue, were identified. Most of the identified metabolites were amino acids, fatty acids, carnitines, lysoglycerophospholipids, sphingomyelins, plasmalogens and glycerophospholipids. Moreover, metabolites related to N-acyl ethanolamine (NAE) biosynthesis, namely glycerophospho (N-acyl) ethanolamines (GP-NAE), which discriminated early-stage SCC from ADC, have also been identified. On the other hand, the analysis of plasma of chronic obstructive pulmonary disease (COPD) and NSCLC patients allowed exclusion of the metabolites related to the inflammatory state in lungs and the identification of compounds (lysoglycerophospholipids, glycerophospholipids and sphingomyelins) truly characteristic to cancer. Our results, among already known, showed novel, thus far not described, metabolites discriminating NSCLC subtypes, especially in the early stage of cancer. Moreover, the presented results also indicated the activity of new metabolic pathways in NSCLC. Further investigations on the role of NAE biosynthesis pathways in the early stage of NSCLC may reveal new prognostic and diagnostic targets.

Metabolomics Reveals Differences in Aqueous Humor Composition in Patients With and Without Pseudoexfoliation Syndrome.

Pseudoexfoliation syndrome (XFS) is stress- or inflammation-induced elastosis accompanied by excessive production of microfibrils and their deposition in the anterior segment of the eye. Approximately 60-70 million people are affected by XFS worldwide. It is a component of a systemic disorder, considered a major risk factor for accelerated cataract formation, cataract surgery complications and development of glaucoma, which untreated or inadequately treated may lead to blindness. Moreover, XFS has been associated with cardiovascular and cerebrovascular morbidity, dementia, sensorineural hearing loss and pelvic organ prolapse. The pathogenesis of XFS has not been fully elucidated yet. Aqueous humor (AH) is a transparent fluid filling the anterior and posterior chambers of the eye. Determination of AH metabolites that are characteristic for XFS may provide valuable information about the molecular background of this ocular disorder. The aim of this study was to compare the composition of AH in XFS and non-XFS patients undergoing cataract surgery. The AH samples from 34 patients (15 with XFS and 19 without) were analyzed using liquid chromatography coupled to a Quadrupole Time-of-Flight mass spectrometer (LC-QTOF-MS). The obtained metabolic fingerprints were analyzed using multivariate statistics. Eleven statistically significant metabolites were identified. Compared with the non-XFS group, the AH of patients with XFS contained significantly lower levels of amino acids and their derivatives, for example, arginine (-31%, VIP = 2.38) and homo-arginine (-19%, VIP = 1.38). Also, a decrease in the levels of two acylcarnitines, hydroxybutyrylcarnitine (-29%, VIP = 1.24) and decatrienoylcarnitine (-46%, VIP = 1.89), was observed. However, the level of indoleacetaldehyde in XFS patients was significantly higher (+96%, VIP = 2.64). Other significant metabolites were two well-recognized antioxidants, ascorbic acid (-33%, VIP = 2.11) and hydroxyanthranilic acid (-33%, VIP = 2.25), as well as S-adenosylmethionine, a compound with anti-inflammatory properties (-29%, VIP = 1.93). Metabolic pathway analysis demonstrated that the identified metabolites belonged to eight metabolic pathways, with cysteine and methionine metabolism as well as arginine and proline metabolism being the most frequently represented. XFS can be associated with enhanced oxidative stress and inflammation, as well as with the disturbances of cellular respiration and mitochondrial energy production. Implementation of non-targeted metabolomics provided a better insight into the still not fully understood pathogenesis of XFS.

Untargeted Metabolomics and Inflammatory Markers Profiling in Children With Crohn's Disease and Ulcerative Colitis-A Preliminary Study.

BACKGROUND: Metabolic profiling might be used to identify disease biomarkers. The aim of our study was to determine the usefulness of untargeted metabolomics analysis to detect differences in serum metabolites between newly diagnosed and untreated pediatric patients with Crohn's disease (CD) or ulcerative colitis (UC) in comparison with a control group (Ctr). Moreover, we investigated the potential of profiling metabolomics and inflammatory markers to improve the noninvasive diagnosis of CD and UC in children. METHODS: Metabolic fingerprinting of serum samples was estimated with liquid chromatography coupled with mass spectrometry in children with CD (n = 9; median age, 14 years), UC (n = 10; median age, 13.5 years), and controls (n = 10; median age, 12.5 years). RESULTS: The majority of chemically annotated metabolites belonged to phospholipids and were downregulated in CD and UC compared with the Ctr. Only 1 metabolite, lactosylceramide 18:1/16:0 (LacCer 18:1/16:0), significantly discriminated CD from UC patients. Interestingly, combining LacCer 18:1/16:0 with other inflammatory markers resulted in a significant increase in the area under the curve with the highest specificity and sensitivity. CONCLUSIONS: Using serum untargeted metabolomics, we have shown that LacCer 18:1/16:0 is a very unique metabolite for CD patients.

An exploratory LC-MS-based metabolomics study reveals differences in aqueous humor composition between diabetic and non-diabetic patients with cataract.

Cataract is the leading cause of blindness worldwide. Epidemiological studies revealed up to a fivefold increased prevalence of cataracts in diabetic subjects. Metabolomics is nowadays frequently implemented to understand pathophysiological processes responsible for disease occurrence and progression. It has also been used recently to study the metabolic composition of aqueous humor (AH). AH is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. The aim of this study was to use metabolomics to compare the AH of diabetic and non-diabetic patients undergoing cataract surgery. Several antioxidants (methyltetrahydrofolic acid, taurine, niacinamide, xanthine, and uric acid) were found decreased (-22 to -61%, p-value 0.05-0.003) in AH of diabetics. Also amino acids (AA) and derivatives were found decreased (-21 to -36%, p-value 0.05-0.01) while glycosylated AA increased (+75-98%, p-value 0.03-0.009) in this group of patients. Metformin was detected in AH of people taking this drug. To our knowledge, this is the first metabolomics study aiming to assess differences in AH composition between diabetic and non-diabetic patients with cataract. An increased oxidative stress and perturbations in amino acid metabolism in AH may be responsible for earlier cataract onset in diabetic patients.

Development of LC-QTOF-MS method for human lung tissue fingerprinting. A preliminary application to nonsmall cell lung cancer.

The major histologic subtypes of non-small cell lung cancer (NSCLC) include adenocarcinoma (ADC), squamous cell lung carcinoma (SCC), and large-cell carcinoma (LCC). Clinical trials of targeted agents and newer chemotherapy agents yielded differences in outcomes according to histologic subgroups providing a rationale for histology-based treatment in NSCLC. Currently, NSCLC subtyping is performed based on histopathological examinations and immunohistochemistry. However available methods leave about 10% of NSCLC cases as not otherwise specified. The purpose of this study was development of an LC-QTOF-MS method for human lung tissue metabolic fingerprinting that could discriminate NSCLC histological subtypes and propose biomarkers candidates that could support proper NSCLC diagnosis. Metabolites were extracted with acetonitrile or methanol/ethanol and different chromatographic conditions were tested. In the final method 10 mg of lung tissue was homogenized with 50% methanol and metabolites were extracted with acetonitrile. Metabolites were separated on C8-RP and HILIC columns. About 3500 and 2000 of metabolic features (in both ion modes) were detected with good repeatability (CV < 20%) by RP and HILIC methods, respectively. Lung tumor and control tissue samples obtained from NSCLC patients were analyzed with developed methodology. Acylcarnitines, fatty acids, phospholipids, and amino acids were found more abundant in tumor as compared to control tissue. Acylcarnitines, lysophospholipids, creatinine, creatine, and alanine were identified as potential targets enabling classification of NSCLC subtypes.