The Relationship between Mitochondrial Respiratory Chain Activities in Muscle and Metabolites in Plasma and Urine: A Retrospective Study

Mitochondria are essential organelles present in eukaryotic cells. They perform vital roles in many cellular pathways; however, their main function is to supply cellular energy in the form of adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS) performed by the mitochondrial respiratory chain (MRC). The MRC located in mitochondrial inner membrane is comprised of ninety proteins organized into five multi-subunit enzymatic protein complexes. These proteins and many other auxiliary proteins are essential for maintaining MRC, and OXPHOS is encoded by two genomes, the nuclear genome and the mitochondrial genome (mtDNA). Consequences of OXPHOS dysfunction include not only energy (ATP) depletion but also elevated oxidative stress, disturbed mitochondrial membrane potential, subsequently leading to imbalanced calcium homeostasis, autophagy/mycophagy, apoptosis, and ultimately to cell death. Mitochondrial diseases affecting one or more MRC complexes are common (prevalence 5 to 15 cases per 100,000 individuals), disabling, progressive, or fatal disorders affecting several vital organs including the brain, optic nerves, the liver, skeletal muscles, and the heart, manifesting at birth, during infancy, and in adulthood [1,2,3]. They are mostly caused by mutations in genes leading to dysfunction of one or multiple MRCs or auxiliary proteins crucial for OXPHOS but could also be secondary to other conditions [4]. The extreme heterogeneity of mitochondrial disorders makes diagnosis complex and sometimes requires the investigation of MRC function in the affected tissues. Before an invasive procedure such as muscle biopsy is performed, a metabolic workup is usually performed in blood and/or urine. Exome sequencing has recently become a valuable diagnostic tool; however, the interpretation is complex when numerous pathogenic and/or new variants are detected [1,2,3,5]. In this retrospective study, we investigated the relationship between common metabolic tests and MRC dysfunction detected in muscle in order to facilitate the diagnostic workup of mitochondrial diseases.

The purpose of this study was to retrospectively examine the correlation between biochemical findings and MRC enzymatic activity in an unbiased manner. Obviously, this study is limited as it includes neither clinical (not reported in a systematic manner) nor molecular data (not available for most cases; however, from our limited knowledge, we estimate that at least 20% of the cases are molecularly defined). We made an effort, but were unable to establish correlations between the clinical symptoms and the different groups. We kindly refer to the literature for this complex issue [1,2,3,4,5]. Moreover, only data obtained from muscle are presented since we did not have pertinent data for liver tissue; therefore, conditions such as certain mtDNA depletion syndromes with variable tissue expression could have been overlooked in this study [9]. The limit of less than 50% residual activity normalized to citrate synthase was determined to potentially include cases of mtDNA heteroplasmy, and, from our experience, this cutoff is relevant [10]. On the other hand, a partial deficiency could also be secondary [4] to other conditions linked or apparently not linked to MRC function, as exemplified by decreased cytochrome c oxidase activity in Troyer syndrome [11], thus the inclusion criteria is indeed a complex, unresolved issue. Additionally, we did not include cases with pyruvate dehydrogenase E1 or E3 deficiency. Nevertheless, this study provides some pertinent information that could facilitate the decision of how to proceed with the workup of a patient clinically suspected to harbor a mitochondrial disease, and provides a complement to other studies [1]. According to the presented data, plasma amino acids did not disclose a significant correlation with MRC dysfunction as most showed a normal profile while a small proportion disclosed mainly elevated Alanine without significant correlation with plasma LA. We also did not detect any specific correlation with Citrulline and/or Arginine, as has been previously reported in MELAS [12], the reason for this, could be that our cohort did not include this specific condition. According to our data, acylcarnine test is also not very informative. Theoretically, impaired MRC should affect mitochondrial fatty acid oxidation with subsequent accumulation of acylcarnitine; however, we mostly detected signs of ketosis or decreased free carnitine. These findings are in accord with the consensus reported by the Mitochondrial Medicine Society [5]. Obviously, if indicated, both amino acid and acylcarnitine tests are important to rule out urea cycle disorders, amino acid degradation defects, fatty acid oxidation defects, primary/secondary carnitine deficiency, etc. and should therefore not be neglected. It is also anticipated that patients exhibiting abnormal acylcarnitine or amino acids, patterns consistent with a known inborn error of metabolism, would not be referred for a muscle biopsy. Still in the context of mitochondrial diseases urinary organic acid analysis seems to be considerably more informative, as the four out of five groups of MRC defects disclosed abnormally elevated excretion of one of several metabolites. Moreover, it was possibly to link certain patterns with certain MRC defects. LA and ketones, mostly concurrently, were prevalent in CI and combined defects. Notably, the combined defects encompass combinations of CI,III,IV,V defects, while complex II, which is solely encoded by the nuclear genome, is normal, so this group includes both mtDNA depletion and translation defects [1,9,13]. 3MGA aciduria is relatively prevalent in CIV and CV defect and is among several other disorders, been linked TMEM70 mutations in CV [14]. Recently elevated urinary 3MGA levels were reported in mitochondrial membrane defects with or without enzymatic MRC dysfunction, thus 3MGA remains an important biomarker for mitochondrial disease [15,16]. Although quantitative measurement in urine was not performed, elevated urine and LA were, as expected, closely correlated, serving as an additional marker of hyper lactic acidemia >6–10 mM [17]. Elevated 4-hydroxyphenyllactate, a Tyrosine metabolite in urine, and Tyrosine in plasma are also associated with liver dysfunction [18]. In this respect, the organic acid analysis is more sensitive as a marker since many cases showed increased 4-hydroxyphenyllactate while only a few had elevated Tyrosine. The more prevalent occurrence of urinary tyrosine metabolites in CI and CIV indicate liver involvement in the pathogenesis of these conditions. The significant correlation between abnormal MRC activities and plasma FGF21 confirms that this protein is a good marker for MRC dysfunction in accord with previously reported findings [19]. Moreover, the finding that elevated FGF21 was prevalent in the combined group and suspected of mitochondrial translation or maintenance defects is certainly consistent with the recent report by Lehtonen et al. [20]. Nevertheless, as some discrepancies were observed between plasma FGF21 and urine organic acids, it seems that the combination of these two tests would be more informative than each one alone. As we have previously pointed out, this study is limited, and there are several other biochemical parameters that were not included in this because of a lack of sufficient data. Among these are growth differentiation factor 15, amino acids, liver function tests, pyruvate, muscle pathology, etc. [5,20,21]. Nevertheless, according to our findings, testing patients suspected of a mitochondrial diseases, for organic acids in urine and FGF21 in plasma is informative and the results facilitate the decision whether to perform a biopsy or not. Alternatively, or in addition, these tests could also be useful in guiding the filtering of variants in exome analysis.

We conclude that urine organic acid test in combination with plasma FGF21 determination are valuable tools in the diagnosis of mitochondrial diseases.