Our biochemical characterization of methylmalonic acidemia in murine and human systems has provided us with important new insights not examined in studies of a different murine model of methylmalonic acidemia [45
]. For example, during gestation (E19) the plasma MMA of muto
mice was in the 170 μM range, much greater than the 30 μM concentration seen in amniotic fluid (urine) samples from mothers known to carry muto
]. These high plasma MMA levels indicate aberrant prenatal metabolism and suggest that the animals may be born partly intoxicated due to the chronicity and magnitude of their exposure to MMA and related metabolites. Prenatal exposure to an abnormal metabolic milieu may explain the onset of clinical symptoms in some of the neonates with muto
methylmalonic acidemia in the first 24 hours of life and the later presentations seen in patients with less severe blocks[4
]. In utero
metabolic changes and the effects on growth and development have become an increasingly recognized feature in disorders of the electron transport chain [47
] and future studies of in utero
metabolism in the muto
mice will examine whether the massive metabolite elevations observed have physiological consequences on energy metabolism.
The pattern of organ MMA concentrations observed as the illness progresses in mice reveals several important trends that can be directly correlated to patients. While the mice are alive and feeding, their plasma MMA concentration is 1.2 mM and the skeletal muscle contains the highest average concentration of MMA per milligram of wet-weight tissue. This suggests that the skeletal muscle is generating significant amounts of MMA. The notion of extra-hepatorenal production of MMA is supported by the studies on the human combined liver-kidney transplant patients, who have massive MMA output (Fig. ) and enormously elevated plasma MMA levels comparable to those seen in non-transplanted methylmalonic acidemia patients. Furthermore, because both liver-kidney transplant patients had intact renal function and normal-functioning livers that would metabolize gut-derived propionate, the MMA in these patients must originate principally in other organs.
Direct comparison to the pre-transplant state in solid organ transplant patients was not possible because they had not been studied when renal function was intact nor in the same fashion described in this report. For this reason, the transplant patients were compared to a group of untransplanted patients with the same enzymatic phenotype and preserved renal function who were treated with low protein, high-energy diets. Future efforts to characterize the metabolic parameters in MMA solid organ transplant recipients might benefit from standardized pre- and post- operative regimens to measure metabolic parameters in all patients. Clearly, this will be an important clinical and research problem to address, especially since the number of MMA transplant recipients to date is small but likely to increase as more patients are identified through newborn screening.
In humans, approximately 40 percent of the body mass is skeletal muscle, which plays a major role in branched-chain amino acid catabolism and fatty acid oxidation [48
]. Because branched-chain amino acid catabolism and odd-chained fatty acid oxidation produce the bulk of the MMA observed in non-transplanted patients[52
], the simplest hypothesis to unify the observations from the murine and human studies is that MMA is being generated by the skeletal muscles in both species. Indeed, when methylmalonyl-CoA mutase was previously measured in different murine tissues, skeletal muscle possessed significant holo- and apo- enzyme activity. Furthermore, the promoter was more complex than seen in generic "house-keeping" genes and the Mut gene and protein appeared to be subject to significant regulation at the transcriptional and post-translational levels [53
]. To directly examine the skeletal muscle and other tissues for the relative abundance of enzyme, we used Western blot analysis to examine tissue extracts from a wild type mouse and found that the skeletal muscles contained a large amount of immunoreactive enzyme, grossly comparable to the relative amounts present in the kidney and liver (see additional file 6
for a Western analysis of murine tissues). Thus several lines of evidence indicate that the enzyme is expressed, active and abundant in the skeletal muscle.
Skeletal muscle is also an important site of branched-chain amino acid (BCAA) oxidation [50
], a process suspected to contribute approximately 50 percent of the MMA load observed in humans [52
]. The branched-chain alpha-ketoacid dehydrogenase complex is an enzyme involved in branch-chain aminoacid oxidation, which is inhibited via phosphorylation by the branched-chain alpha-ketoacid dehydrogenase kinase [54
]. Methylmalonyl-CoA has previously been shown to inhibit branched-chain alpha-ketoacid dehydrogenase kinase activity [55
]. In the setting of methylmalonic acidemia, methylmalonyl-CoA accretion may increase the activity of the branched-chain alpha-ketoacid dehydrogenase complex by inhibiting the branched-chain alpha-ketoacid dehydrogenase kinase in the skeletal muscle beds, setting up a physiological forward feeding circuit for MMA production. Increased MMA production through such a mechanism might also contribute to the significant methylmalonic acidemia seen in the transplant patients. The possibility that the MMA concentrations in the tissues of the knock-out mice may be influenced by the developmental regulation for BCAA catabolism will require further examination, especially since the regulation of BCAA catabolism in mice has not been extensively studied. Additionally, the precursors of MMA may vary over time, with odd-chained fatty acids potentially providing a major contribution to the metabolite load in the mice and humans, especially given the evidence from patient studies demonstrating that odd chained fatty acids are increased in patients with propionate metabolic disorders[56
The clinical studies presented here include determination of whole body MMA output in muto
patients, a measurement that has previously been demonstrated to be an effective means to carefully describe whole body MMA metabolism[44
]. Previous clinical reports [33
] and the studies presented here firmly demonstrate that enzymatic correction of the liver and kidney in affected patients by transplantation does not always greatly lower the circulating MMA load. However, liver transplantation patients do realize complete protection from systemic metabolic decompensation. This suggests a tissue-specific response or maladaption to the inherent biochemical lesion in the native liver, and possibly other organs. Our results indicate that modulating metabolism in the skeletal muscle – by gene replacement or pharmacological means – may represent an effective strategy to lower MMA levels and increase metabolic capacity in affected patients. Other disorders of intermediary metabolism that feature impaired branched chain aminoacid oxidation, such as propionic acidemia, might also benefit from these approaches.