Effect of an eight-week high-intensity interval training programme on circulating sphingolipid levels in middle-aged adults at elevated cardiometabolic risk (SphingoFIT)-Protocol for a randomised controlled exercise trial.

INTRODUCTION: Evidence indicates that sphingolipid accumulation drives complex molecular alterations promoting cardiometabolic diseases. Clinically, it was shown that sphingolipids predict cardiometabolic risk independently of and beyond traditional biomarkers such as low-density lipoprotein cholesterol. To date, little is known about therapeutic modalities to lower sphingolipid levels. Exercise, a powerful means to prevent and treat cardiometabolic diseases, is a promising modality to mitigate sphingolipid levels in a cost-effective, safe, and patient-empowering manner. METHODS: This randomised controlled trial will explore whether and to what extent an 8-week fitness-enhancing training programme can lower serum sphingolipid levels of middle-aged adults at elevated cardiometabolic risk (n = 98, 50% females). The exercise intervention will consist of supervised high-intensity interval training (three sessions weekly), while the control group will receive physical activity counselling based on current guidelines. Blood will be sampled early in the morning in a fasted state before and after the 8-week programme. Participants will be provided with individualised, pre-packaged meals for the two days preceding blood sampling to minimise potential confounding. An 'omic-scale sphingolipid profiling, using high-coverage reversed-phase liquid chromatography coupled to tandem mass spectrometry, will be applied to capture the circulating sphingolipidome. Maximal cardiopulmonary exercise tests will be performed before and after the 8-week programme to assess patient fitness changes. Cholesterol, triglycerides, glycated haemoglobin, the homeostatic model assessment for insulin resistance, static retinal vessel analysis, flow-mediated dilatation, and strain analysis of the heart cavities will also be assessed pre- and post-intervention. This study shall inform whether and to what extent exercise can be used as an evidence-based treatment to lower circulating sphingolipid levels. TRIAL REGISTRATION: The trial was registered on www.clinicaltrials.gov (NCT06024291) on August 28, 2023.

Molecular insights into sex-specific metabolic alterations in Alzheimer's mouse brain using multi-omics approach.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by altered cellular metabolism in the brain. Several of these alterations have been found to be exacerbated in females, known to be disproportionately affected by AD. We aimed to unravel metabolic alterations in AD at the metabolic pathway level and evaluate whether they are sex-specific through integrative metabolomic, lipidomic, and proteomic analysis of mouse brain tissue. METHODS: We analyzed male and female triple-transgenic mouse whole brain tissue by untargeted mass spectrometry-based methods to obtain a molecular signature consisting of polar metabolite, complex lipid, and protein data. These data were analyzed using multi-omics factor analysis. Pathway-level alterations were identified through joint pathway enrichment analysis or by separately evaluating lipid ontology and known proteins related to lipid metabolism. RESULTS: Our analysis revealed significant AD-associated and in part sex-specific alterations across the molecular signature. Sex-dependent alterations were identified in GABA synthesis, arginine biosynthesis, and in alanine, aspartate, and glutamate metabolism. AD-associated alterations involving lipids were also found in the fatty acid elongation pathway and lysophospholipid metabolism, with a significant sex-specific effect for the latter. CONCLUSIONS: Through multi-omics analysis, we report AD-associated and sex-specific metabolic alterations in the AD brain involving lysophospholipid and amino acid metabolism. These findings contribute to the characterization of the AD phenotype at the molecular level while considering the effect of sex, an overlooked yet determinant metabolic variable.

Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

The fate of orally administered sialic acid: First insights from patients with N-acetylneuraminic acid synthase deficiency and control subjects.

BACKGROUND: In NANS deficiency, biallelic mutations in the N-acetylneuraminic acid synthase (NANS) gene impair the endogenous synthesis of sialic acid (N-acetylneuraminic acid) leading to accumulation of the precursor, N-acetyl mannosamine (ManNAc), and to a multisystemic disorder with intellectual disability. The aim of this study was to determine whether sialic acid supplementation might be a therapeutic avenue for NANS-deficient patients. METHODS: Four adults and two children with NANS deficiency and four adult controls received oral NeuNAc acid (150 mg/kg/d) over three days. Total NeuNAc, free NeuNAc and ManNAc were analyzed in plasma and urine at different time points. RESULTS: Upon NeuNAc administration, plasma free NeuNAc increased within hours (P < 0.001) in control and in NANS-deficient individuals. Total and free NeuNAc concentrations also increased in the urine as soon as 6 h after beginning of oral administration in both groups. NeuNAc did not affect plasma and urinary ManNAc, that remained higher in NANS deficient subjects than in controls (day 1-3; all P < 0.01). Oral NeuNAc was well tolerated with no significant side effects. DISCUSSION: Orally administered free NeuNAc was rapidly absorbed but also rapidly excreted in the urine. It did not change ManNAc levels in either patients or controls, indicating that it may not achieve enough feedback inhibition to reduce ManNAc accumulation in NANS-deficient subjects. Within the limitations of this study these results do not support a potential for oral free NeuNAc in the treatment of NANS deficiency but they provide a basis for further therapeutic approaches in this condition.

Metabolic View on Human Healthspan: A Lipidome-Wide Association Study.

As ageing is a major risk factor for the development of non-communicable diseases, extending healthspan has become a medical and societal necessity. Precise lipid phenotyping that captures metabolic individuality could support healthspan extension strategies. This study applied 'omic-scale lipid profiling to characterise sex-specific age-related differences in the serum lipidome composition of healthy humans. A subset of the COmPLETE-Health study, composed of 73 young (25.2 ± 2.6 years, 43% female) and 77 aged (73.5 ± 2.3 years, 48% female) clinically healthy individuals, was investigated, using an untargeted liquid chromatography high-resolution mass spectrometry approach. Compared to their younger counterparts, aged females and males exhibited significant higher levels in 138 and 107 lipid species representing 15 and 13 distinct subclasses, respectively. Percentage of difference ranged from 5.8% to 61.7% (females) and from 5.3% to 46.0% (males), with sphingolipid and glycerophophospholipid species displaying the greatest amplitudes. Remarkably, specific sphingolipid and glycerophospholipid species, previously described as cardiometabolically favourable, were found elevated in aged individuals. Furthermore, specific ether-glycerophospholipid and lyso-glycerophosphocholine species displayed higher levels in aged females only, revealing a more favourable lipidome evolution in females. Altogether, age determined the circulating lipidome composition, while lipid species analysis revealed additional findings that were not observed at the subclass level.

Sex-specific alterations in NAD+ metabolism in 3xTg Alzheimer's disease mouse brain assessed by quantitative targeted LC-MS.

Levels of nicotinamide adenine dinucleotide (NAD+) are known to decline with age and have been associated with impaired mitochondrial function leading to neurodegeneration, a key facet of Alzheimer's disease (AD). NAD+synthesis is sustained via tryptophan-kynurenine (Trp-Kyn) pathway as de novo synthesis route, and salvage pathways dependent on the availability of nicotinic acid and nicotinamide. While being currently investigated as a multifactorial disease with a strong metabolic component, AD remains without curative treatment and important sex differences were reported in relation to disease onset and progression. The aim of this study was to reveal the potential deregulation of NAD+metabolism in AD with the direct analysis of NAD+precursors in the mouse brain tissue (wild type (WT) versus triple transgenic (3xTg) AD), using a sex-balanced design. To this end, we developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the measurement of the full spectrum of NAD+precursors and intermediates in all three pathways. In brain tissue of mice with developed AD symptoms, a decrease in kynurenine (Kyn) versus increase in kynurenic acid (KA) levels were observed in both sexes with a significantly higher increment of KA in males. These alterations in Trp-Kyn pathway might be a consequence of neuroinflammation and a compensatory production of neuroprotective kynurenic acid. In the NAD+ salvage pathway, significantly lower levels of nicotinamide mononucleotide (NMN) were measured in the AD brain of males and females. Depletion of NMN implies the deregulation of salvage pathway critical for maintaining optimal NAD+ levels and mitochondrial and neuronal function.

Metabolic Impairment in Coronary Artery Disease: Elevated Serum Acylcarnitines Under the Spotlights.

Coronary artery disease (CAD) remains the leading cause of death worldwide. Expanding patients' metabolic phenotyping beyond clinical chemistry investigations could lead to earlier recognition of disease onset and better prevention strategies. Additionally, metabolic phenotyping, at the molecular species level, contributes to unravel the roles of metabolites in disease development. In this cross-sectional study, we investigated clinically healthy individuals (n = 116, 65% male, 70.8 ± 8.7 years) and patients with CAD (n = 54, 91% male, 67.0 ± 11.5 years) of the COmPLETE study. We applied a high-coverage quantitative liquid chromatography-mass spectrometry approach to acquire a comprehensive profile of serum acylcarnitines, free carnitine and branched-chain amino acids (BCAAs), as markers of mitochondrial health and energy homeostasis. Multivariable linear regression analyses, adjusted for confounders, were conducted to assess associations between metabolites and CAD phenotype. In total, 20 short-, medium- and long-chain acylcarnitine species, along with L-carnitine, valine and isoleucine were found to be significantly (adjusted p ≤ 0.05) and positively associated with CAD. For 17 acylcarnitine species, associations became stronger as the number of affected coronary arteries increased. This implies that circulating acylcarnitine levels reflect CAD severity and might play a role in future patients' stratification strategies. Altogether, CAD is characterized by elevated serum acylcarnitine and BCAA levels, which indicates mitochondrial imbalance between fatty acid and glucose oxidation.

Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome.

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.

Metabolomics meets lipidomics: Assessing the small molecule component of metabolism.

Metabolomics, including lipidomics, is emerging as a quantitative biology approach for the assessment of energy flow through metabolism and information flow through metabolic signaling; thus, providing novel insights into metabolism and its regulation, in health, healthy ageing and disease. In this forward-looking review we provide an overview on the origins of metabolomics, on its role in this postgenomic era of biochemistry and its application to investigate metabolite role and (bio)activity, from model systems to human population studies. We present the challenges inherent to this analytical science, and approaches and modes of analysis that are used to resolve, characterize and measure the infinite chemical diversity contained in the metabolome (including lipidome) of complex biological matrices. In the current outbreak of metabolic diseases such as cardiometabolic disorders, cancer and neurodegenerative diseases, metabolomics appears to be ideally situated for the investigation of disease pathophysiology from a metabolite perspective.

Systemic and central nervous system metabolic alterations in Alzheimer's disease.

BACKGROUND: Metabolic alterations, related to cerebral glucose metabolism, brain insulin resistance, and age-induced mitochondrial dysfunction, play an important role in Alzheimer's disease (AD) on both the systemic and central nervous system level. To study the extent and significance of these alterations in AD, quantitative metabolomics was applied to plasma and cerebrospinal fluid (CSF) from clinically well-characterized AD patients and cognitively healthy control subjects. The observed metabolic alterations were associated with core pathological processes of AD to investigate their relation with amyloid pathology and tau-related neurodegeneration. METHODS: In a case-control study of clinical and biomarker-confirmed AD patients (n = 40) and cognitively healthy controls without cerebral AD pathology (n = 34) with paired plasma and CSF samples, we performed metabolic profiling, i.e., untargeted metabolomics and targeted quantification. Targeted quantification focused on identified deregulated pathways highlighted in the untargeted assay, i.e. the TCA cycle, and its anaplerotic pathways, as well as the neuroactive tryptophan and kynurenine pathway. RESULTS: Concentrations of several TCA cycle and beta-oxidation intermediates were higher in plasma of AD patients, whilst amino acid concentrations were significantly lower. Similar alterations in these energy metabolism intermediates were observed in CSF, together with higher concentrations of creatinine, which were strongly correlated with blood-brain barrier permeability. Alterations of several amino acids were associated with CSF Amyloidß1-42. The tryptophan catabolites, kynurenic acid and quinolinic acid, showed significantly higher concentrations in CSF of AD patients, which, together with other tryptophan pathway intermediates, were correlated with either CSF Amyloidß1-42, or tau and phosphorylated Tau-181. CONCLUSIONS: This study revealed AD-associated systemic dysregulation of nutrient sensing and oxidation and CNS-specific alterations in the neuroactive tryptophan pathway and (phospho)creatine degradation. The specific association of amino acids and tryptophan catabolites with AD CSF biomarkers suggests a close relationship with core AD pathology. Our findings warrant validation in independent, larger cohort studies as well as further investigation of factors such as gender and APOE genotype, as well as of other groups, such as preclinical AD, to identify metabolic alterations as potential intervention targets.

Merged Targeted Quantification and Untargeted Profiling for Comprehensive Assessment of Acylcarnitine and Amino Acid Metabolism.

Acylcarnitines and amino acids are key players in energy metabolism; however, analytical methods for comprehensive and straightforward quantitative profiling of these metabolites, without derivatization or use of ion-pairing agents, are lacking. We therefore developed a hydrophilic interaction chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the simultaneous quantification of acylcarnitines and amino acids in a single run, while taking advantage of HRMS data acquired in full-scan mode to screen for additional derivatives and other polar metabolites. A single-step metabolite extraction with internal standard mixture (in methanol) warranted high-throughput sample preparation whose applicability was demonstrated on a panel of human biofluids (i.e., blood plasma, CSF, and urine) and brain tissue. Method accuracy was within 90-106% of validated NIST reference plasma concentrations for the panel of measured amino acids. Amino acid and acylcarnitine extraction recoveries were 87-100% on average, depending on the concentration range spiked. The coefficient of variation (CV) was 1-10% and 1-25% for intra- and interday measurements, respectively, with the highest CVs for the metabolites at the limit of quantification, depending on the biofluid. Acylcarnitine and amino acid signatures or chemical composition barcodes of the different biofluids and human brain tissue were acquired and biofluid- and tissue-associated differences were discussed in the context of their respective physiological roles. Significant differences were observed in the amino acid profiles, whereas acylcarnitine composition did not show biofluid-characteristic or brain region-specific pattern. The retrospective exploration of full-scan all-ion-fragmentation data allowed us to extract the information on unsaturated and hydroxylated acylcarnitine species, amines, and purine and pyrimidine metabolites. This merged targeted and untargeted approach provides an innovative strategy for simultaneous and comprehensive assessment of acylcarnitine and amino acid metabolism in clinical research studies using relevant biofluids and tissue extracts.

LC-HRMS data as a result of untargeted metabolomic profiling of human cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF is analyzed in the context of neurological diseases and is rarely collected from healthy subjects. For this reason, the metabolite variation associated with general phenotypic characteristics such as gender and age have hardly ever been studied. Here we present the hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) data as a result of untargeted metabolomics analysis of a cohort of elderly cognitively healthy volunteers (n = 32). 146 unambiguously identified water soluble metabolites (using accurate mass, retention time and MS/MS matching against spectral libraries) were measured and their abundances across all the subjects depending on their gender are provided in this article. Data tables are available at https://data.mendeley.com/datasets/c73xtsd4s5/1. it's published on mendeley, the DOI is DOI:10.17632/c73xtsd4s5.1. The data presented in this article are related to the research article entitled "A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects" (Gallart-Ayala et al., 2018, In press).

A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF has been profiled in the context of neurological diseases to provide novel insights into the disease mechanisms. However, a global high-throughput approach to measure a broad diversity of polar metabolites present in CSF is lacking. Although still perceived as challenging and less reproducible, hydrophilic interaction liquid chromatography (HILIC) has recently evolved to offer the unprecedented coverage capacity of water-soluble metabolome. Here, we present a global HILIC high-resolution mass spectrometry-based (HRMS) approach that combines the profiling in acidic pH ESI (+) and basic pH ESI (-) mode to extend the coverage of CSF polar metabolome. This approach allowed us to annotate and measure a broad range of central carbon metabolites (implicated in glycolysis, TCA cycle, nucleotide, amino acid and fatty acid metabolism) in CSF collected from cognitively healthy elderly volunteers (n = 32), using a single extraction method. Metabolite annotation was achieved using the accurate mass, RT and MS/MS criteria, allowing for the characterization of 146 measurable metabolites. Exploration of characterized individual CSF profiles allowed for a discovery of intriguing gender-associated differences, with significantly higher acylcarnitine levels in men and higher taurine levels women. With this case study, we demonstrate the value of combined HILIC ESI ± HRMS profiling to assess CSF metabolome in clinical research studies.

XCMS-MRM and METLIN-MRM: a cloud library and public resource for targeted analysis of small molecules.

We report XCMS-MRM and METLIN-MRM ( http://xcmsonline-mrm.scripps.edu/ and http://metlin.scripps.edu/ ), a cloud-based data-analysis platform and a public multiple-reaction monitoring (MRM) transition repository for small-molecule quantitative tandem mass spectrometry. This platform provides MRM transitions for more than 15,500 molecules and facilitates data sharing across different instruments and laboratories.

New in vitro model derived from brain-specific Mut-/- mice confirms cerebral ammonium accumulation in methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria (MMAuria) is an inborn error of metabolism leading to neurological deterioration. In this study, we used 3D organotypic brain cell cultures derived from embryos of a brain-specific Mut-/- (brain KO) mouse to investigate mechanisms leading to brain damage. We challenged our in vitro model by a catabolic stress (temperature shift). RESULTS: Typical metabolites for MMAuria as well as a massive NH4+ increase were found in the media of brain KO cultures. We investigated different pathways of intracerebral NH4+ production and found increased expression of glutaminase 2 and diminished expression of GDH1 in Mut-/- aggregates. While all brain cell types appeared affected in their morphological development in Mut-/- aggregates, the most pronounced effects were observed on astrocytes showing swollen fibers and cell bodies. Inhibited axonal elongation and delayed myelination of oligodendrocytes were also noted. Most effects were even more pronounced after 48 h at 39 °C. Microglia activation and an increased apoptosis rate suggested degeneration of Mut-/- brain cells. NH4+ accumulation might be the trigger for all observed alterations. We also found a generalized increase of chemokine concentrations in Mut-/- culture media at an early developmental stage followed by a decrease at a later stage. CONCLUSION: We proved for the first time that Mut-/- brain cells are indeed able to produce the characteristic metabolites of MMAuria. We confirmed significant NH4+ accumulation in culture media of Mut-/- aggregates, suggesting that intracellular NH4+ concentrations might even be higher, gave first clues on the mechanisms leading to NH4+ accumulation in Mut-/- brain cells, and showed the involvement of neuroinflammatory processes in the neuropathophysiology of MMAuria.

α-ketoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming.

Glutamine metabolism provides synergistic support for macrophage activation and elicitation of desirable immune responses; however, the underlying mechanisms regulated by glutamine metabolism to orchestrate macrophage activation remain unclear. Here we show that the production of α-ketoglutarate (αKG) via glutaminolysis is important for alternative (M2) activation of macrophages, including engagement of fatty acid oxidation (FAO) and Jmjd3-dependent epigenetic reprogramming of M2 genes. This M2-promoting mechanism is further modulated by a high αKG/succinate ratio, whereas a low ratio strengthens the proinflammatory phenotype in classically activated (M1) macrophages. As such, αKG contributes to endotoxin tolerance after M1 activation. This study reveals new mechanistic regulations by which glutamine metabolism tailors the immune responses of macrophages through metabolic and epigenetic reprogramming.

Role of sulfotransferases in resveratrol metabolism in human adipocytes.

SCOPE: Polyphenols such as resveratrol received interest for their wide-ranging biological benefits, including anti-obesity potential, mimicking effects of caloric restriction with reduced body fat and increased energy expenditure. However, resveratrol is rapidly metabolized, and it is not completely understood which form of resveratrol is responsible for the effects observed within target cells such as adipocytes. Also the role of metabolizing enzymes has not been investigated before. METHODS AND RESULTS: Resveratrol metabolism was evaluated in human adipocytes by UHPLC-MS at low physiological doses. Resveratrol was found to rapidly metabolize into its sulfated form, while resveratrol glucuronides were undetectable. Only resveratrol, but not its sulfated nor glucuronidated forms had an antilipolytic effect on adipocytes. The metabolizing enzyme responsible for sulfation of polyphenols is SULT1A1, and was found to be upregulated upon adipocyte differentiation. Knocking down SULT1A1 in adipocytes led to decreased resveratrol sulfate and increased resveratrol intra- and extracellularly. This lower SULT1A1 activity resulted in an increased antilipolytic effect of resveratrol on adipocytes, as demonstrated by lower glycerol accumulation, which could be attributed to lower activity of the lipolytic protein, perilipin. CONCLUSION: Sulfotransferase activity modulates metabolism of resveratrol in adipocytes with potential consequences on bioavailability and thus metabolic action of this polyphenol.