Defining functional classes of Barth syndrome mutation in humans

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ

Principles and practice of lipidomics

The technical advances in mass spectrometry, particularly the development of (ultra)-high-resolution/mass accuracy measurement capabilities in combination with refinement of soft ionization techniques, have increased the application and success of lipidomics to answer biological questions in relation to lipid metabolism. Together with other omics technologies, lipidomics has become an important tool to practice systems biology as lipids comprise a very significant part of the metabolome and play pleiotropic roles in cellular functions. As an increasing number of disorders are linked to lipid metabolism, lipidomics is used to search for biomarkers, understand disease mechanism and follow the efficacy of therapeutic options. This review provides a first introduction to the major methodological strategies currently used for mass spectrometry-based lipidomics and associated data pre-processing and analysi

MetaboShiny: interactive analysis and metabolite annotation of mass spectrometry-based metabolomics data

Direct infusion untargeted mass spectrometry-based metabolomics allows for rapid insight into a sample’s metabolic activity. However, analysis is often complicated by the large array of detected m/z values and the difficulty to prioritize important m/z and simultaneously annotate their putative identities. To address this challenge, we developed MetaboShiny, a novel R/RShiny-based metabolomics package featuring data analysis, database- and formula-prediction-based annotation and visualization. To demonstrate this, we reproduce and further explore a MetaboLights metabolomics bioinformatics study on lung cancer patient urine samples. MetaboShiny enables rapid and rigorous analysis and interpretation of direct infusion untargeted mass spectrometry-based metabolomics data

Vitamin B6 is essential for serine de novo biosynthesis

Pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation

Plasma lipidomics as a diagnostic tool for peroxisomal disorders

Peroxisomes are ubiquitous cell organelles that play an important role in lipid metabolism. Accordingly, peroxisomal disorders, including the peroxisome biogenesis disorders and peroxisomal single-enzyme deficiencies, are associated with aberrant lipid metabolism. Lipidomics is an emerging tool for diagnosis, disease-monitoring, identifying lipid biomarkers, and studying the underlying pathophysiology in disorders of lipid metabolism. In this study, we demonstrate the potential of lipidomics for the diagnosis of peroxisomal disorders using plasma samples from patients with different types of peroxisomal disorders. We show that the changes in the plasma profiles of phospholipids, di- and triglycerides, and cholesterol esters correspond with the characteristic metabolite abnormalities that are currently used in the metabolic screening for peroxisomal disorders. The lipidomics approach, however, gives a much more detailed overview of the metabolic changes that occur in the lipidome. Furthermore, we identified novel unique lipid species for specific peroxisomal diseases that are candidate biomarkers. The results presented in this paper show the power of lipidomics approaches to enable the specific diagnosis of different peroxisomal disorder

Functional characterisation of peroxisomal β-oxidation disorders in fibroblasts using lipidomics

Peroxisomes play an important role in a variety of metabolic pathways, including the α- and β-oxidation of fatty acids, and the biosynthesis of ether phospholipids. Single peroxisomal enzyme deficiencies (PEDs) are a group of peroxisomal disorders in which either a peroxisomal matrix enzyme or a peroxisomal membrane transporter protein is deficient. To investigate the functional consequences of specific enzyme deficiencies on the lipidome, we performed lipidomics using cultured skin fibroblasts with different defects in the β-oxidation of very long-chain fatty acids, including ABCD1- (ALD), acyl-CoA oxidase 1 (ACOX1)-, D-bifunctional protein (DBP)-, and acyl-CoA binding domain containing protein 5 (ACBD5)-deficient cell lines. Ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry revealed characteristic changes in the phospholipid composition in fibroblasts with different fatty acid β-oxidation defects. Remarkably, we found that ether phospholipids, including plasmalogens, were decreased. We defined specific phospholipid ratios reflecting the different enzyme defects, which can be used to discriminate the PED fibroblasts from healthy control cells

Aqueous Humor Analysis Identifies Higher Branched Chain Amino Acid Metabolism as a Marker for Human Leukocyte Antigen-B27 Acute Anterior Uveitis and Disease Activity

Purpose: Human leukocyte antigen-B27 (HLA-B27)–positive acute anterior uveitis (AAU) has a higher recurrence rate and shows more anterior chamber cell infiltration compared with HLA-B27–negative patients, suggesting distinct etiologies of these clinically overlapping conditions. To advance our understanding of the biology of AAU, we characterized the metabolic profile of aqueous humor (AqH) of patients with HLA-B27–associated AAU (B27-AAU) and noninfectious idiopathic AAU (idiopathic AAU). Design: Experimental laboratory study. Methods: AqH samples from 2 independent cohorts totaling 30 patients with B27-AAU, 16 patients with idiopathic AAU, and 20 patients with cataracts underwent 2 individual rounds of direct infusion mass spectrometry. Features predicted by direct infusion mass spectrometry that facilitated maximum separation between the disease groups in regression models were validated by liquid chromatography/tandem mass spectrometry–based quantification with appropriate standards. Results: Partial least square-discriminant analysis revealed metabolite profiles that were able to separate patients with B27-AAU from those with iodiopathic AAU. Pathway enrichment analysis, based on metabolites on which separation of the groups in the partial least square-discriminant analysis model was based, demonstrated the involvement of branched-chain amino acid biosynthesis, ascorbate and aldarate metabolism, the tricarboxylic acid cycle, and glycolysis-diverting pathways (eg, serine biosynthesis) across all investigated cohorts. Notably, the metabolite ketoleucine was elevated in B27-AAU across all 3 runs and moderately—but robustly—correlated with anterior chamber cell count (correlation coefficient range 0.41–0.81). Conclusions: These results illustrate metabolic heterogeneity between HLA-B27–positive and HLA-B27–negative AAU, including an increase of branched-chain amino acid biosynthesis, that reflects disease activity in AAU

Time-restricted feeding during the inactive phase abolishes the daily rhythm in mitochondrial respiration in rat skeletal muscle

Shift-workers show an increased incidence of type 2 diabetes mellitus (T2DM). A possible mechanism is the disruption of the circadian timing of glucose homeostasis. Skeletal muscle mitochondrial function is modulated by the molecular clock. We used time-restricted feeding (TRF) during the inactive phase to investigate how mistimed feeding affects muscle mitochondrial metabolism. Rats on an ad libitum (AL) diet were compared to those that could eat only during the light (inactive) or dark (active) phase. Mitochondrial respiration, metabolic gene expressions, and metabolite concentrations were determined in the soleus muscle. Rats on AL feeding or dark-fed TRF showed a clear daily rhythm in muscle mitochondrial respiration. This rhythm in mitochondrial oxidative phosphorylation capacity was abolished in light-fed TRF animals and overall 24h respiration was lower. The expression of several genes involved in mitochondrial biogenesis and the fission/fusion machinery was altered in light-fed animals. Metabolomics analysis indicated that light-fed animals had lost rhythmic levels of α-ketoglutarate and citric acid. Contrastingly, lipidomics showed that light-fed animals abundantly gained rhythmicity in levels of triglycerides. Furthermore, while the RER shifted entirely with the food intake in the light-fed animals, many measured metabolic parameters (e.g., activity and mitochondrial respiration) did not strictly align with the shifted timing of food intake, resulting in a mismatch between expected metabolic supply/demand (as dictated by the circadian timing system and light/dark-cycle) and the actual metabolic supply/demand (as dictated by the timing of food intake). These data suggest that shift-work impairs mitochondrial metabolism and causes metabolic inflexibility, which can predispose to T2DM

Time-restricted feeding during the inactive phase abolishes the daily rhythm in mitochondrial respiration in rat skeletal muscle

Shift-workers show an increased incidence of type 2 diabetes mellitus (T2DM). A possible mechanism is the disruption of the circadian timing of glucose homeostasis. Skeletal muscle mitochondrial function is modulated by the molecular clock. We used time-restricted feeding (TRF) during the inactive phase to investigate how mistimed feeding affects muscle mitochondrial metabolism. Rats on an ad libitum (AL) diet were compared to those that could eat only during the light (inactive) or dark (active) phase. Mitochondrial respiration, metabolic gene expressions, and metabolite concentrations were determined in the soleus muscle. Rats on AL feeding or dark-fed TRF showed a clear daily rhythm in muscle mitochondrial respiration. This rhythm in mitochondrial oxidative phosphorylation capacity was abolished in light-fed TRF animals and overall 24h respiration was lower. The expression of several genes involved in mitochondrial biogenesis and the fission/fusion machinery was altered in light-fed animals. Metabolomics analysis indicated that light-fed animals had lost rhythmic levels of α-ketoglutarate and citric acid. Contrastingly, lipidomics showed that light-fed animals abundantly gained rhythmicity in levels of triglycerides. Furthermore, while the RER shifted entirely with the food intake in the light-fed animals, many measured metabolic parameters (e.g., activity and mitochondrial respiration) did not strictly align with the shifted timing of food intake, resulting in a mismatch between expected metabolic supply/demand (as dictated by the circadian timing system and light/dark-cycle) and the actual metabolic supply/demand (as dictated by the timing of food intake). These data suggest that shift-work impairs mitochondrial metabolism and causes metabolic inflexibility, which can predispose to T2DM