Untargeted Metabolomics Analysis Reveals a Link between ETHE1-Mediated Disruptive Redox State and Altered Metabolic Regulation

Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography–mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD⁺ and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder

A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption

The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC–HR-TOFMS). The aim of this study was to investigate whether such UPLC–HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers’ positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, <i>S</i>-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC–HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology

Infarct size and correlation with blood glucose.

<p><b>A.</b> Infarct size/area-at-risk ratio (IS/AAR). Black bars: Control, White bars: ZDF. <b>B.</b> Correlations between preoperative fasting blood glucose and IS/AAR at prediabetes, onset and late type 2 diabetes mellitus. Mean ± SEM. Circles: ZDF 6 weeks, Squares: ZDF 12 weeks, Triangles: ZDF 24 weeks.</p

Myocardial glycogen concentrations.

<p>Myocardial concentrations of total-glycogen in vivo (<b>A</b>) and after ischemia and 30 min reperfusion (<b>B</b>) in control and ZDF rats. Mean ± SEM. Black bars: Control, White bars: ZDF. <b>IR</b>: ischemia-reperfusion. p<0.0001 vs. age-matched control and ZDF 6 weeks.</p

Study design and experimental protocol.

<p>Overview of groups, number of animals (<b>N</b>) and experimental protocols. <b>KH-buffer:</b> Krebs-Henseleit buffer.</p

Expression of proteins involved in the malate-aspartate shuttle.

<p>Fold change in expression of proteins participating in the malate-aspartate shuttle. Controls are set to 0 at all ages. Bars indicate fold change in ZDF hearts compared to control hearts. <b>A:</b> Transporter proteins. <b>B:</b> Enzymes. C: Representative western blots. There were no significant changes in expression at 6 and 12 weeks, while citrin and EAAT1 were significantly, and aralar non-significantly, downregulated at 24 weeks of age. Mean ± SEM. <b>EAAT</b>: Excitatory Amino Acid Transporter. <b>ASAT:</b> Aspartate Amino Acid Transaminase. <b>MDH:</b> Malate Dehydrogenase. *p<0.05.</p

Animal characteristics.

<p>Mean±SEM.</p>*<p>p<0.05 compared to age-matched controls.</p>†<p>p<0.05 compared to 6 weeks control/ZDF.</p

Interstitial concentrations of metabolites.

<p>Interstitial concentrations of glycolytic - and citric acid cycle metabolites measured by myocardial microdialysis during ischemia-reperfusion in control (closed squares) and ZDF (open squares) at 12 weeks. Mean ± SEM. <b>A:</b> Lactate, <b>B:</b> Pyruvate, <b>C:</b> Succinate, <b>D:</b> Fumarate, <b>E:</b> Malate.</p

Glucose metabolism prior to ischemia-reperfusion.

<p>Glucose uptake – glycolysis- and glucose oxidation rates during stabilization. Mean ± SEM. Black bars: Control, White bars: ZDF.</p

Glucose metabolism during reperfusion.

<p><b>A</b>: Glycolysis and <b>B:</b> Glucose oxidation rates during reperfusion at 6, 12 and 24 weeks. Mean ± SEM. Closed symbols: Control, Open symbols: ZDF. P-values indicate differences between control and ZDF from 0–5 and 10–30 minutes of reperfusion, respectively.</p