High-throughput assay to measure oxygen consumption in digitonin-permeabilized cells of patients with mitochondrial disorders.
Clin Chem
; 56(3): 424-31, 2010 Mar.
Article
in En
| MEDLINE
| ID: mdl-20044447
ABSTRACT
BACKGROUND:
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.METHODS:
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.RESULTS:
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.CONCLUSIONS:
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Oxygen Consumption
/
Mitochondrial Diseases
/
High-Throughput Screening Assays
Type of study:
Diagnostic_studies
Limits:
Humans
Language:
En
Journal:
Clin Chem
Journal subject:
QUIMICA CLINICA
Year:
2010
Document type:
Article
Affiliation country:
Netherlands