Abnormal tricarboxylic acid cycle metabolites in isovaleric acidaemia

Summary Background  Although a number of abnormal diagnostic metabolites have previously been described in the urine of patients with isovaleric acidaemia (IVA), they do not fully explain the clinical symptoms associated with this disease. Methods  On the basis of our current understanding of the TCA cycle and IVA, we predicted a number of abnormal methylated TCA cycle metabolites, initiated by methylsuccinic acid. We subsequently obtained characteristic gas chromatography–mass spectrometry elution times and mass spectra of the chemically synthesized predicted compounds and screened the urine of 6 IVA patients and 24 age-matched controls. Further proof for our findings was generated from a series of in vitro enzyme reactions using the chemically synthesized standards as substrates to their respective TCA cycle enzymes. Results  Apart from the previously described methylsuccinic and methylfumaric acid, 3-methylmalic acid, (2R,3S)- and (2R,3R)-methylcitric acid and 2-methyl-cis-aconitic acid were detected in the urine of all 6 IVA patients in increased amounts. Additionally, although not directly determined, the in vitro enzyme reaction using of 3-methylmalic acid and malate dehydrogenase, in conjunction with the detection of 2-ketobutyric acid in the urine of all 6 IVA patients, strongly suggests an additional synthesis of 3-methyloxaloacetic acid by the same cycle. Conclusion  Not only do these newly identified metabolites serve as additional diagnostic markers to those previously identified in IVA, but due to the structural arrangements of the (2R,3R)-methylcitric acid and 2-methyl-cis-aconitinic acid-derived 2-methylisocitric acid, inhibition of normal TCA cycle metabolism results at citrate synthase and isocitrate dehydrogenase, respectively. Synopsis  Methylsuccinic acid acts as the initiating substrate to a series of abnormal, potentially harmful, methylated tricarboxylic acid cycle metabolites in isovaleric acidaemia. Competing interests: None declared References to electronic databases: Isovaleric acidaemia: OMIM #243500. Isovaleryl-CoA dehydrogenase: EC 1.3.99.10.