Propionic acidemia (PA) and methylmalonic acidemia (MMA) are autosomal recessive disorders of propionyl-CoA (P-CoA) catabolism, which are caused by a deficiency in the enzyme propionyl-CoA carboxylase or the enzyme methylmalonyl-CoA (MM-CoA) mutase, respectively. The functional consequence of PA or MMA is the inability to catabolize P-CoA to MM-CoA or MM-CoA to succinyl-CoA, resulting in the accumulation of P-CoA and other metabolic intermediates, such as propionylcarnitine (C3), 3-hydroxypropionic acid, methylcitric acid (MCA), and methylmalonic acid (only in MMA). P-CoA and its metabolic intermediates, at high concentrations found in PA and MMA, inhibit enzymes in the first steps of the urea cycle as well as enzymes in the tricarboxylic acid (TCA) cycle, causing a reduction in mitochondrial energy production. We previously showed that metabolic defects of PA could be recapitulated using PA patient-derived primary hepatocytes in a novel organotypic system. Here, we sought to investigate whether treatment of normal human primary hepatocytes with propionate would recapitulate some of the biochemical features of PA and MMA in the same platform. We found that high levels of propionate resulted in high levels of intracellular P-CoA in normal hepatocytes. Analysis of TCA cycle intermediates by GC–MS/MS indicated that propionate may inhibit enzymes of the TCA cycle as shown in PA, but is also incorporated in the TCA cycle, which does not occur in PA. To better recapitulate the disease phenotype, we obtained hepatocytes derived from livers of PA and MMA patients. We characterized the PA and MMA donors by measuring key proximal biomarkers, including P-CoA, MM-CoA, as well as clinical biomarkers propionylcarnitine-to-acetylcarnitine ratios (C3/C2), MCA, and methylmalonic acid. Additionally, we used isotopically-labeled amino acids to investigate the contribution of relevant amino acids to production of P-CoA in models of metabolic stability or acute metabolic crisis. As observed clinically, we demonstrated that the isoleucine and valine catabolism pathways are the greatest sources of P-CoA in PA and MMA donor cells and that each donor showed differential sensitivity to isoleucine and valine. We also studied the effects of disodium citrate, an anaplerotic therapy, which resulted in a significant increase in the absolute concentration of TCA cycle intermediates, which is in agreement with the benefit observed clinically. Our human cell-based PA and MMA disease models can inform preclinical drug discovery and development where mouse models of these diseases are inaccurate, particularly in well-described species differences in branched-chain amino acid catabolism.

丙酸血症（PA）和甲基丙二酸血症（MMA）是丙酰辅酶A（P-CoA）分解代谢的常染色体隐性遗传疾病，其是由丙酰辅酶A羧化酶或甲基丙二酰辅酶A（MM-CoA）酶缺乏引起的突变。PA或MMA的功能后果是无法将P-CoA分解代谢为MM-CoA或MM-CoA分解为琥珀酰-CoA，导致P-CoA和其他代谢中间体（如丙酰肉碱（C3），3-羟基丙酸，甲基柠檬酸（MCA）和甲基丙二酸（仅在MMA中）。在PA和MMA中发现高浓度的P-CoA及其代谢中间体会抑制尿素循环第一步中的酶以及三羧酸（TCA）循环中的酶，从而降低线粒体能量的产生。我们以前表明，在新的器官型系统中，使用PA患者衍生的原代肝细胞可以概括PA的代谢缺陷。在这里，我们试图研究用丙酸酯处理正常人原代肝细胞是否会在同一平台上概括PA和MMA的某些生化特征。我们发现高水平的丙酸导致正常肝细胞中高水平的细胞内P-CoA。通过GC-MS / MS分析TCA循环中间体表明，丙酸可能会抑制PAA循环中的酶，如PA中所示，但也掺入了TCA循环中，PA中不存在。为了更好地概括疾病表型，我们获得了来自PA和MMA患者肝脏的肝细胞。我们通过测量关键的近端生物标志物（包括P-CoA）对PA和MMA供体进行了表征，MM-CoA以及临床生物标记丙酰肉碱与乙酰肉碱的比率（C3 / C2），MCA和甲基丙二酸。此外，我们使用同位素标记的氨基酸来研究代谢稳定性或急性代谢危机模型中相关氨基酸对P-CoA产生的贡献。如临床观察到的，我们证明了异亮氨酸和缬氨酸的分解代谢途径是PA和MMA供体细胞中P-CoA的最大来源，并且每个供体对异亮氨酸和缬氨酸的敏感性不同。我们还研究了枸ap酸二钠（一种过继疗法）的作用，该作用导致TCA循环中间体的绝对浓度显着增加，这与临床观察到的益处一致。