OBJECTIVE: This study aims to characterize the metabolic alterations in patients with inherited mitochondrial enzymopathies. We focused on wide-coverage targeted metabolomic, organic acid and lipidomic analyses of patients with TMEM70 deficiency (TMEM70d), short-chain acyl-CoA dehydrogenase deficiency (SCADd), and individuals with both deficiencies (TMEM70d-SCADd). METHODS: Serum and urine samples were collected from patients with TMEM70d (n = 13), SCADd (n = 11), TMEM70d-SCADd (n = 3), and controls (n = 38). Analyses were conducted using high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Univariate and multivariate statistical evaluation was performed to identify significant metabolic differences between patient groups and controls. RESULTS: Distinct metabolic profiles were observed in urine and serum samples of patients with TMEM70d, SCADd, and TMEM70d-SCADd compared to controls. Urinary metabolomics revealed significant elevations in butyrylcarnitine and metabolites related to branched-chain amino acid degradation in SCADd and TMEM70d-SCADd patients. Serum metabolomic analysis indicated alterations in pyruvate metabolism, citric acid cycle intermediates, and acylcarnitine metabolism in TMEM70d and TMEM70d-SCADd patients. Lipidomic analysis showed decreased levels of glycerophospholipids and sphingolipids across all patient groups. CONCLUSION: Patients with TMEM70d, SCADd, and TMEM70d-SCADd exhibit distinct metabolic signatures characterized by disturbances in energy metabolism, amino acid degradation, and lipid homeostasis. The combination of TMEM70d and SCADd leads to synergistic metabolic effects, emphasizing the importance of comprehensive metabolic profiling in understanding complex mitochondrial disorders and identifying potential biomarkers for diagnosis and treatment monitoring.

2nd Faculty of Medicine Charles University Praha Czech Republic

Department of Clinical Biochemistry University Hospital Olomouc Czech Republic

Department of Laboratory Medicine Centre for Inherited Metabolic Disorders National Institute of Children's Diseases Bratislava Slovak Republic

Department of Pediatric and Adolescent Medicine Faculty of Medicine Pavol Josef Safarik University Košice Slovak Republic

Department of Pediatrics Centre for Inherited Metabolic Disorders National Institute of Children's Diseases Bratislava Slovak Republic

Laboratory for Inherited Metabolic Disorders Faculty of Medicine and Dentistry Palacký University Olomouc Olomouc Czech Republic

Metabolic Outpatient Clinic Children's Faculty Hospital Košice Slovak Republic

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Galber C., Carissimi S., Baracca A., Giorgio V. The ATP synthase deficiency in human diseases. Life. 2021;11 doi: 10.3390/life11040325. PubMed DOI PMC

Carroll J., He J., Ding S., Fearnley I.M., Walker J.E. TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I. Proc. Natl. Acad. Sci. U. S. A. 2021;118 doi: 10.1073/pnas.2100558118. PubMed DOI PMC

Hejzlarová K., Tesařová M., Vrbacká-Čížková A., Vrbacký M., Hartmannová H., Kaplanová V., et al. Expression and processing of the TMEM70 protein. Biochim. Biophys. Acta. 2011;1807:144–149. PubMed

Houstek J., Mrácek T., Vojtísková A., Zeman J. Mitochondrial diseases and ATPase defects of nuclear origin. Biochim. Biophys. Acta. 2004;1658:115–121. PubMed

Cízková A., Stránecký V., Mayr J.A., Tesarová M., Havlícková V., Paul J., et al. TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy. Nat. Genet. 2008;40:1288–1290. PubMed

Magner M., Dvorakova V., Tesarova M., Mazurova S., Hansikova H., Zahorec M., et al. TMEM70 deficiency: long-term outcome of 48 patients. J. Inherit. Metab. Dis. 2015;38:417–426. PubMed

Torraco A., Verrigni D., Rizza T., Meschini M.C., Vazquez-Memije M.E., Martinelli D., et al. TMEM70: a mutational hot spot in nuclear ATP synthase deficiency with a pivotal role in complex V biogenesis. Neurogenetics. 2012;13:375–386. PubMed

Kovalčíkova J., Vrbacký M., Pecina P., Tauchmannová K., Nůsková H., Kaplanová V., et al. TMEM70 facilitates biogenesis of mammalian ATP synthase by promoting subunit c incorporation into the rotor structure of the enzyme. FASEB J. 2019;33:14103–14117. PubMed

Baban A., Adorisio R., Corica B., Rizzo C., Calì F., Semeraro M., et al. Delayed appearance of 3-methylglutaconic aciduria in neonates with early onset metabolic cardiomyopathies: a potential pitfall for the diagnosis. Am. J. Med. Genet. A. 2020;182:64–70. PubMed

Lin Y., Zhang W., Chen D., Lin C., Zheng Z., Fu Q., et al. Newborn screening and genetic characteristics of patients with short- and very long-chain acyl-CoA dehydrogenase deficiencies. Clin. Chim. Acta. 2020;510:285–290. PubMed

Gallant N.M., Leydiker K., Tang H., Feuchtbaum L., Lorey F., Puckett R., et al. Biochemical, molecular, and clinical characteristics of children with short chain acyl-CoA dehydrogenase deficiency detected by newborn screening in California. Mol. Genet. Metabol. 2012;106:55–61. PubMed

Nochi Z., Olsen R.K.J., Gregersen N. Short-chain acyl-CoA dehydrogenase deficiency: from gene to cell pathology and possible disease mechanisms. J. Inherit. Metab. Dis. 2017;40:641–655. PubMed

Pedersen C.B., Kølvraa S., Kølvraa A., Stenbroen V., Kjeldsen M., Ensenauer R., et al. The ACADS gene variation spectrum in 114 patients with short-chain acyl-CoA dehydrogenase (SCAD) deficiency is dominated by missense variations leading to protein misfolding at the cellular level. Hum. Genet. 2008;124:43–56. PubMed

Waisbren S.E., Levy H.L., Noble M., Matern D., Gregersen N., Pasley K., et al. Short-chain acyl-CoA dehydrogenase (SCAD) deficiency: an examination of the medical and neurodevelopmental characteristics of 14 cases identified through newborn screening or clinical symptoms. Mol. Genet. Metabol. 2008;95:39–45. PubMed PMC

Lisyová J., Chandoga J., Jungová P., Repiský M., Knapková M., Machková M., et al. An unusually high frequency of SCAD deficiency caused by two pathogenic variants in the ACADS gene and its relationship to the ethnic structure in Slovakia. BMC Med. Genet. 2018;19:64. PubMed PMC

Yuan Y., Yang S., Deng D., Chen Y., Zhang C., Zhou R., et al. Effects of genetic variations in Acads gene on the risk of chronic obstructive pulmonary disease. IUBMB Life. 2020;72:1986–1996. PubMed

Arnold G.L. Inborn errors of metabolism in the 21 century: past to present. Ann. Transl. Med. 2018;6:467. PubMed PMC

van Maldegem B.T., Wanders R.J.A., Wijburg F.A. Clinical aspects of short-chain acyl-CoA dehydrogenase deficiency. J. Inherit. Metab. Dis. 2010;33:507–511. PubMed PMC

Amendt B.A., Greene C., Sweetman L., Cloherty J., Shih V., Moon A., et al. Short-chain acyl-coenzyme A dehydrogenase deficiency. Clinical and biochemical studies in two patients. J. Clin. Investig. 1987;79:1303–1309. PubMed PMC

Tonin R., Caciotti A., Funghini S., Pasquini E., Mooney S.D., Cai B., et al. Clinical relevance of short-chain acyl-CoA dehydrogenase (SCAD) deficiency: exploring the role of new variants including the first SCAD-disease-causing allele carrying a synonymous mutation. BBA Clin. 2016;5:114–119. PubMed PMC

Piskláková B., Friedecká J., Ivanovová E., Hlídková E., Bekárek V., Prídavok M., et al. Rapid and efficient LC-MS/MS diagnosis of inherited metabolic disorders: a semi-automated workflow for analysis of organic acids, acylglycines, and acylcarnitines in urine. Clin. Chem. Lab. Med. 2023 doi: 10.1515/cclm-2023-0084. PubMed DOI

Körver-Keularts I.M.L.W., Wang P., Waterval H.W.A.H., Kluijtmans L.A.J., Wevers R.A., Langhans C.-D., et al. Fast and accurate quantitative organic acid analysis with LC-QTOF/MS facilitates screening of patients for inborn errors of metabolism. J. Inherit. Metab. Dis. 2018;41:415–424. PubMed PMC

Cífková E., Brumarová R., Ovčačíková M., Dobešová D., Mičová K., Kvasnička A., et al. Lipidomic and metabolomic analysis reveals changes in biochemical pathways for non-small cell lung cancer tissues. Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2022;1867 PubMed

Kvasnička A., Friedecký D., Brumarová R., Pavlíková M., Pavelcová K., Mašínová J., et al. Alterations in lipidome profiles distinguish early-onset hyperuricemia, gout, and the effect of urate-lowering treatment. Arthritis Res. Ther. 2023;25:234. PubMed PMC

AlzbetaG. AlzbetaG/Metabol. First version. Zenodo; 2019. DOI

Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D., et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. PubMed PMC

Barschak A.G., Ferreira G.C., André K.R., Schuck P.F., Viegas C.M., Tonin A., et al. Inhibition of the electron transport chain and creatine kinase activity by ethylmalonic acid in human skeletal muscle. Metab. Brain Dis. 2006;21:11–19. PubMed

Honzík T., Tesarová M., Mayr J.A., Hansíková H., Jesina P., Bodamer O., et al. Mitochondrial encephalocardio-myopathy with early neonatal onset due to TMEM70 mutation. Arch. Dis. Child. 2010;95:296–301. PubMed

Staretz-Chacham O., Wormser O., Manor E., Birk O.S., Ferreira C.R. TMEM70 deficiency: novel mutation and hypercitrullinemia during metabolic decompensation. Am. J. Med. Genet. A. 2019;179:1293–1298. PubMed PMC

Esterhuizen K., van der Westhuizen F.H., Louw R. Metabolomics of mitochondrial disease. Mitochondrion. 2017;35:97–110. PubMed

Chen Q., Kirk K., Shurubor Y.I., Zhao D., Arreguin A.J., Shahi I., et al. Rewiring of glutamine metabolism is a bioenergetic adaptation of human cells with mitochondrial DNA mutations. Cell Metab. 2018;27:1007–1025. e5. PubMed PMC

Holness M.J., Sugden M.C. Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem. Soc. Trans. 2003;31:1143–1151. PubMed

Eaton S. Control of mitochondrial beta-oxidation flux. Prog. Lipid Res. 2002;41:197–239. PubMed

Vianey-Liaud C., Divry P., Gregersen N., Mathieu M. The inborn errors of mitochondrial fatty acid oxidation. J. Inherit. Metab. Dis. 1987;10(Suppl 1):159–200. PubMed

Lazarow P.B., De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc. Natl. Acad. Sci. U. S. A. 1976;73:2043–2046. PubMed PMC

Vissing C.R., Dunø M., Wibrand F., Christensen M., Vissing J. Hydroxylated long-chain acylcarnitines are biomarkers of mitochondrial myopathy. J. Clin. Endocrinol. Metab. 2019;104:5968–5976. PubMed

Larson A.A., Balasubramaniam S., Christodoulou J., Burrage L.C., Marom R., Graham B.H., et al. Biochemical signatures mimicking multiple carboxylase deficiency in children with mutations in MT-ATP6. Mitochondrion. 2019;44:58–64. PubMed PMC

Legault J.T., Strittmatter L., Tardif J., Sharma R., Tremblay-Vaillancourt V., Aubut C., et al. A metabolic signature of mitochondrial dysfunction revealed through a monogenic form of Leigh syndrome. Cell Rep. 2015;13:981–989. PubMed PMC

Roe D.S., Yang B.Z., Vianey-Saban C., Struys E., Sweetman L., Roe C.R. Differentiation of long-chain fatty acid oxidation disorders using alternative precursors and acylcarnitine profiling in fibroblasts. Mol. Genet. Metabol. 2006;87:40–47. PubMed

Verhoeven N.M., Roe D.S., Kok R.M., Wanders R.J., Jakobs C., Roe C.R. Phytanic acid and pristanic acid are oxidized by sequential peroxisomal and mitochondrial reactions in cultured fibroblasts. J. Lipid Res. 1998;39:66–74. PubMed

Wortmann S.B., Kluijtmans L.A.J., Rodenburg R.J., Sass J.O., Nouws J., van Kaauwen E.P., et al. 3-Methylglutaconic aciduria--lessons from 50 genes and 977 patients. J. Inherit. Metab. Dis. 2013;36:913–921. PubMed

Jones D.E., Perez L., Ryan R.O. 3-Methylglutaric acid in energy metabolism. Clin. Chim. Acta. 2020;502:233–239. PubMed PMC

Su B., Ryan R.O. Metabolic biology of 3-methylglutaconic acid-uria: a new perspective. J. Inherit. Metab. Dis. 2014;37:359–368. PubMed PMC

Duran M., Bruinvis L., Ketting D., Kamerling J.P., Wadman S.K., Schutgens R.B. The identification of (E)-2-methylglutaconic acid, a new isoleucine metabolite, in the urine of patients with beta-ketothiolase deficiency, propionic acidaemia and methylmalonic acidaemia. Biomed. Mass Spectrom. 1982;9:1–5. PubMed

Bao X.R., Ong S.-E., Goldberger O., Peng J., Sharma R., Thompson D.A., et al. Mitochondrial dysfunction remodels one-carbon metabolism in human cells. Elife. 2016;5 doi: 10.7554/eLife.10575. PubMed DOI PMC

Naini A., Kaufmann P., Shanske S., Engelstad K., De Vivo D.C., Schon E.A. Hypocitrullinemia in patients with MELAS: an insight into the “MELAS paradox.”. J. Neurol. Sci. 2005;229–230:187–193. PubMed

Parfait B., de Lonlay P., von Kleist-Retzow J.C., Cormier-Daire V., Chrétien D., Rötig A., et al. The neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G) triggers muscle ATPase deficiency and hypocitrullinaemia. Eur. J. Pediatr. 1999;158:55–58. PubMed

Debray F.-G., Lambert M., Allard P., Mitchell G.A. Low citrulline in Leigh disease: still a biomarker of maternally inherited Leigh syndrome. J. Child Neurol. 2010;25:1000–1002. PubMed

Crippa B.L., Leon E., Calhoun A., Lowichik A., Pasquali M., Longo N. Biochemical abnormalities in Pearson syndrome. Am. J. Med. Genet. A. 2015;167A:621–628. PubMed

van de Poll M.C.G., Ligthart-Melis G.C., Boelens P.G., Deutz N.E.P., van Leeuwen P.A.M., Dejong C.H.C. Intestinal and hepatic metabolism of glutamine and citrulline in humans. J. Physiol. 2007;581:819–827. PubMed PMC

Ligthart-Melis G.C., van de Poll M.C.G., Boelens P.G., Dejong C.H.C., Deutz N.E.P., van Leeuwen P.A.M. Glutamine is an important precursor for de novo synthesis of arginine in humans. Am. J. Clin. Nutr. 2008;87:1282–1289. PubMed

Förstermann U., Closs E.I., Pollock J.S., Nakane M., Schwarz P., Gath I., et al. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension. 1994;23:1121–1131. PubMed

El-Hattab A.W., Emrick L.T., Hsu J.W., Chanprasert S., Almannai M., Craigen W.J., et al. Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation. Mol. Genet. Metabol. 2016;117:407–412. PubMed PMC

Al Jasmi F., Al Zaabi N., Al-Thihli K., Al Teneiji A.M., Hertecant J., El-Hattab A.W. Endothelial dysfunction and the effect of arginine and citrulline supplementation in children and adolescents with mitochondrial diseases. J. Cent. Nerv. Syst. Dis. 2020;12 PubMed PMC

Chester A.H., Yacoub M.H., Moncada S. Nitric oxide and pulmonary arterial hypertension. Glob. Cardiol. Sci. Pract. 2017;2017:14. PubMed PMC

Catteruccia M., Verrigni D., Martinelli D., Torraco A., Agovino T., Bonafé L., et al. Persistent pulmonary arterial hypertension in the newborn (PPHN): a frequent manifestation of TMEM70 defective patients. Mol. Genet. Metabol. 2014;111:353–359. PubMed

Yang X., Liang J., Ding L., Li X., Lam S.-M., Shui G., et al. Phosphatidylserine synthase regulates cellular homeostasis through distinct metabolic mechanisms. PLoS Genet. 2019;15 PubMed PMC

Vankoningsloo S., Piens M., Lecocq C., Gilson A., De Pauw A., Renard P., et al. Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells: role of fatty acid beta-oxidation and glucose. J. Lipid Res. 2005;46:1133–1149. PubMed

Aflaki E., Radovic B., Chandak P.G., Kolb D., Eisenberg T., Ring J., et al. Triacylglycerol accumulation activates the mitochondrial apoptosis pathway in macrophages. J. Biol. Chem. 2011;286:7418–7428. PubMed PMC

Schuck P.F., Busanello E.N.B., Moura A.P., Tonin A.M., Grings M., Ritter L., et al. Promotion of lipid and protein oxidative damage in rat brain by ethylmalonic acid. Neurochem. Res. 2010;35:298–305. PubMed

Schuck P.F., Milanez A.P., Felisberto F., Galant L.S., Machado J.L., Furlanetto C.B., et al. Brain and muscle redox imbalance elicited by acute ethylmalonic acid administration. PLoS One. 2015;10 PubMed PMC

Najdekr L., Gardlo A., Mádrová L., Friedecký D., Janečková H., Correa E.S., et al. Oxidized phosphatidylcholines suggest oxidative stress in patients with medium-chain acyl-CoA dehydrogenase deficiency. Talanta. 2015;139:62–66. PubMed

Wallner S., Schmitz G. Plasmalogens the neglected regulatory and scavenging lipid species. Chem. Phys. Lipids. 2011;164:573–589. PubMed

Luoma A.M., Kuo F., Cakici O., Crowther M.N., Denninger A.R., Avila R.L., et al. Plasmalogen phospholipids protect internodal myelin from oxidative damage. Free Radic. Biol. Med. 2015;84:296–310. PubMed

Katafuchi T., Ifuku M., Mawatari S., Noda M., Miake K., Sugiyama M., et al. Effects of plasmalogens on systemic lipopolysaccharide-induced glial activation and β-amyloid accumulation in adult mice. Ann. N. Y. Acad. Sci. 2012;1262:85–92. PubMed

Dorninger F., Forss-Petter S., Berger J. From peroxisomal disorders to common neurodegenerative diseases - the role of ether phospholipids in the nervous system. FEBS Lett. 2017;591:2761–2788. PubMed PMC

Criscuolo A., Nepachalovich P., Garcia-Del Rio D.F., Lange M., Ni Z., Baroni M., et al. Analytical and computational workflow for in-depth analysis of oxidized complex lipids in blood plasma. Nat. Commun. 2022;13:6547. PubMed PMC