Laboratory of Pediatrics and Neurology, Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands.
Clin Chem. 2010 Mar;56(3):424-31. doi: 10.1373/clinchem.2009.131441. Epub 2009 Dec 31.
Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells.
We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader.
In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells.
These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.
肌肉活检分析被认为是疑似线粒体疾病患者诊断工作的金标准。培养成纤维细胞的分析可以提供重要的额外诊断信息。单个 OXPHOS 复合物的测量并不总是提供有关完整线粒体能量产生系统功能状态的充分信息。因此，我们优化了一种用于患者来源细胞中氧消耗分析的高通量基于荧光的方法。
我们在含有丙酮酸和苹果酸的底物混合物存在下，使用荧光探针、96 孔板和荧光板读数器，分析了二硝基甲烷通透细胞中的线粒体呼吸。
在对照成纤维细胞中，我们观察到 ADP 对丙酮酸+苹果酸驱动呼吸的明显刺激。已知的 OXPHOS 系统和三羧酸循环抑制剂显著降低了呼吸作用。在具有不同 OXPHOS 缺陷的患者成纤维细胞中，与对照细胞相比，ADP 刺激的呼吸活性降低。在一些肌肉组织中 ATP 产生率降低但 OXPHOS 酶活性正常的患者中，成纤维细胞的呼吸活性降低。最后，我们观察到对照组和复合物 I 缺陷的转线粒体细胞之间存在明显差异。
这些结果证实了该测定作为二硝基甲烷通透细胞中线粒体功能高通量筛选方法的有效性。该测定可区分肌肉中的原发性和继发性线粒体异常，这对于咨询非常重要，也将有助于寻找导致线粒体疾病的新遗传缺陷。
