Metabolomics can be described as a simultaneous and comprehensive analysis of small molecules in a biological sample. Recent technological and bioinformatics advances have facilitated large-scale metabolomic studies in many areas, including inborn errors of metabolism (IEMs). Despite significant improvements in the diagnosis and treatment of some IEMs, it is still challenging to understand how genetic variation affects disease progression and susceptibility. In addition, a search for new more personalized therapies and a growing demand for tools to monitor the long-term metabolic effects of existing therapies set the stage for metabolomics integration in preclinical and clinical studies. While targeted metabolomics approach is a common practice in biochemical genetics laboratories for biochemical diagnosis and monitoring of IEMs, applications of untargeted metabolomics in the clinical laboratories are still in infancy, facing some challenges. It is however, expected in the future to dramatically change the scope and utility of the clinical laboratory playing a significant role in patient management. This review provides an overview of targeted and global, large-scale metabolomic studies applied to investigate various IEMs. We discuss an existing and prospective clinical applications of metabolomics in IEMs for better diagnosis and deep understanding of complex metabolic perturbations associated with the etiology of inherited metabolic disorders.

Calorie restriction (CR), an age delaying diet, affects fat oxidation through poorly understood mechanisms. We investigated the effect of CR on fat metabolism gene expression and intermediate metabolites of fatty acid oxidation in the liver. We found that CR changed the liver acylcarnitine profile: acetylcarnitine, short‐chain acylcarnitines, and long‐chain 3‐hydroxy‐acylcarnitines increased, and several long‐chain acylcarnitines decreased. Acetyl‐CoA and short‐chain acyl‐CoAs were also increased in CR. CR did not affect the expression of CPT1 and upregulated the expression of long‐chain and very‐long‐chain Acyl‐CoA dehydrogenases (LCAD and VLCAD, respectively). The expression of downstream enzymes such as mitochondrial trifunctional protein and enzymes in medium‐ and short‐chain acyl‐CoAs oxidation was not affected in CR. CR shifted the balance of fatty acid oxidation enzymes and fatty acid metabolites in the liver. Acetyl‐CoA generated through beta‐oxidation can be used for ketogenesis or energy production. In agreement, blood ketone bodies increased under CR in a time of the day‐dependent manner. Carnitine acetyltransferase (CrAT) is a bidirectional enzyme that interconverts short‐chain acyl‐CoAs and their corresponding acylcarnitines. CrAT expression was induced in CR liver supporting the increased acetylcarnitine and short‐chain acylcarnitine production. Acetylcarnitine can freely travel between cellular sub‐compartments. Supporting this CR increased protein acetylation in the mitochondria, cytoplasm, and nucleus. We hypothesize that changes in acyl‐CoA and acylcarnitine levels help to control energy metabolism and contribute to metabolic flexibility under CR.

Barth syndrome (BTHS) is an X-linked recessive multisystem disorder caused by mutations in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for the acyltransferase tafazzin. This protein is highly expressed in the heart and plays a significant role in cardiolipin biosynthesis. Heart disease is the major clinical manifestation of BTHS with a high incidence in early life. Although the genetic basis of BTHS and tetralinoleoyl cardiolipin deficiency in BTHS-affected individuals are well-established, downstream metabolic changes in cardiac metabolism are still uncovered. Our study aimed to characterize TAZ-induced metabolic perturbations in the heart. Control (PGP1-TAZ^WT) and TAZ mutant (PGP1-TAZ^517delG) iPS-CM were incubated with ¹³C₆-glucose and ¹³C_5-glutamine and incorporation of ¹³C into downstream Krebs cycle intermediates was traced. Our data reveal that TAZ^517delG induces accumulation of cellular long chain acylcarnitines and overexpression of fatty acid binding protein (FABP4). We also demonstrate that TAZ^517delG induces metabolic alterations in pathways related to energy production as reflected by high glucose uptake, an increase in glycolytic lactate production and a decrease in palmitate uptake. Moreover, despite mitochondrial dysfunction, in the absence of glucose and fatty acids, TAZ^517delG-iPS-CM can use glutamine as a carbon source to replenish the Krebs cycle.

Nonalcoholic liver disease (NAFLD) is manifested with a wide spectrum of clinical symptoms and is closely associated with the metabolic syndrome, inflammation, and mitochondrial dysfunction. Although the mechanism of mitochondrial dysfunction in NAFLD is still not fully elucidated, multiple studies have demonstrated evidence of molecular, biochemical, and biophysical mitochondrial abnormalities in NAFLD. Given the association between NAFLD and mitochondrial dysfunction, the aim of this study is to analyze circulating levels of Krebs cycle intermediates in a cohort of NAFLD-affected individuals and matching healthy controls and to correlate our findings with the liver function metrics. Standard serum biochemistry and Krebs cycle intermediates were analyzed in NAFLD (n = 22) and matched control (n = 67) cohorts. Circulating levels of isocitrate and citrate were significantly (p < 0.05) elevated in the NAFLD cohort of patients. The area under the curve (AUROC) for these two metabolites exhibited a moderate clinical utility. Correlations between plasma Krebs cycle intermediates and standard clinical plasma metrics were explored by Pearson’s correlation coefficient. The data obtained for plasma Krebs cycle intermediates suggest pathophysiological insights that link mitochondrial dysfunction with NAFLD. Our findings reveal that plasma isocitrate and citrate can discriminate between normal and NAFLD cohorts and can be utilized as noninvasive markers of mitochondrial dysfunction in NAFLD. Future studies with large populations at different NAFLD stages are warranted.

Inborn errors of metabolism (IEM) represent a group of inherited diseases in which genetic defect leads to the block on a metabolic pathway, resulting in a single enzyme dysfunction. As a downstream consequence of the residual or full loss of the enzymatic activity, there is an accumulation of toxic metabolites in the proximity of the metabolic block and/or a deficiency of an essential metabolic product which leads to the clinical presentation of the disease. While individually IEMs are rare, a collectively estimated incidence of metabolic inherited disorders is 1:800. The genetic basis of IEMs can involve abnormalities such as point mutations, deletions or insertions, or more complex genomic rearrangements. Categorization of IEM can be simply made on the basis of the affected metabolic network: fatty acids oxidation disorders, protein/amino acids metabolism disorders, disorders of carbohydrate metabolism, lysosomal storage diseases, peroxisomal disorders, and mitochondrial diseases. This chapter will overview amino acid metabolism-related inherited disorders and amino acid analysis for the diagnosis and routine monitoring of this category of IEMs.

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD, OMIM 609575) is associated with energy deficiency and mitochondrial dysfunction and may lead to rhabdomyolysis and cardiomyopathy. Under physiological conditions, there is a fine balance between the utilization of different carbon nutrients to maintain the Krebs cycle. The maintenance of steady pools of Krebs cycle intermediates is critical formitochondrial energy homeostasis especially in high-energy demanding organs such as muscle and heart. Even-chain dicarboxylic acids are established as alternative energy carbon sources that replenish the Krebs cycle by bypassing a defective β-oxidation pathway. Despite this, even-chain dicarboxylic acids are eliminated in the urine of VLCAD-affected individuals. In this study, we explore dodecanedioic acid (C12; DODA) supplementation and investigate its metabolic effect on Krebs cycle intermediates, glucose uptake, and acylcarnitine profiles in VLCAD-deficient fibroblasts. Our findings indicate that DODA supplementation replenishes the Krebs cycle by increasing the succinate pool, attenuates glycolytic flux, and reduces levels of toxic very long-chain acylcarnitines.