Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD⁺/NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox‐dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3‐phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD⁺. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-¹³C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD⁺/NADH ratio), consistent with the NAD⁺ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD⁺-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders.

Untargeted metabolomics may become a standard approach to address diagnostic requests, but, at present, data interpretation is very labor-intensive. To facilitate its implementation in metabolic diagnostic screening, we developed a method for automated data interpretation that preselects the most likely inborn errors of metabolism (IEM). The input parameters of the knowledge-based algorithm were (1) weight scores assigned to 268 unique metabolites for 119 different IEM based on literature and expert opinion, and (2) metabolite Z-scores and ranks based on direct-infusion high resolution mass spectrometry. The output was a ranked list of differential diagnoses (DD) per sample. The algorithm was first optimized using a training set of 110 dried blood spots (DBS) comprising 23 different IEM and 86 plasma samples comprising 21 different IEM. Further optimization was performed using a set of 96 DBS consisting of 53 different IEM. The diagnostic value was validated in a set of 115 plasma samples, which included 58 different IEM and resulted in the correct diagnosis being included in the DD of 72% of the samples, comprising 44 different IEM. The median length of the DD was 10 IEM, and the correct diagnosis ranked first in 37% of the samples. Here, we demonstrate the accuracy of the diagnostic algorithm in preselecting the most likely IEM, based on the untargeted metabolomics of a single sample. We show, as a proof of principle, that automated data interpretation has the potential to facilitate the implementation of untargeted metabolomics for metabolic diagnostic screening, and we provide suggestions for further optimization of the algorithm to improve diagnostic accuracy.

Pain often persists in patients with inflammatory diseases, even when the inflammation has subsided. The molecular mechanisms leading to this failure in resolution of inflammatory pain and the transition to chronic pain are poorly understood. Mitochondrial dysfunction in sensory neurons has been linked to chronic pain, but its role in resolution of inflammatory pain is unclear. Transient inflammation causes neuronal plasticity, called hyperalgesic priming, which impairs resolution of hyperalgesia induced by a subsequent inflammatory stimulus. We identified that hyperalgesic priming in mice caused disturbances in mitochondrial respiration, oxidative stress, and redox balance in dorsal root ganglia (DRG) neurons. Preventing these priming-induced disturbances restored resolution of inflammatory hyperalgesia. Concurrent with these mitochondrial and metabolic changes, the expression of ATPSc-KMT, a mitochondrial methyltransferase, was increased in DRG neurons in primed mice. ATPSc-KMT overexpression in DRG neurons of naive mice induced similar mitochondrial and metabolic changes as observed after priming, leading to failure in pain resolution. Inhibition of mitochondrial respiration, knockdown of ATPSCKMT expression, or NAD ⁺ supplementation were sufficient to restore resolution of inflammatory pain and prevent chronic pain development. Thus, inflammation-induced mitochondrial-dependent disturbances in DRG neurons promote failure in inflammatory pain resolution and drive the transition to chronic pain.