KWANTIFISERING VAN MITOCHONDRIALE RESPIRASIE OM AANGEBORE MITOCHONDRIALE DEFEKTE TE ONDERSOEK

Abstract

Genetic defects of oxidative phosphorylation account for a large variety of clinical symptoms in childhood. The mitochondrial respiratory chain catalyses the oxidation of fuel molecules with the concomitant energy transduction into ATP. During the oxidation process electrons are transferred to oxygen via the energy transducing complexes of the respiratory chain. When a defect occurs in one of these complexes of the respiratory chain, the electrons can not be transferred through that complex and less electrons are transferred to oxygen and consequently less ATP is produced. The diagnosis of respiratory chain deficiencies are difficult because the complexes are coded for by both chromosomal and mitochondrial DNA. The Metabolic Laboratory of the Biochemistry and Microbiology department at the PU for CHE is involved in the diagnoses and detection of inherited metabolic defects, therefor there is a need for a standardised method with which an inherited metabolic defect can be traced in patients. Many different methods for the quantification of mitochondrial respiration were examined. Mitochondrial respiration can for example be measured with polarography as a function of the amount of oxygen used or the amount of ATP produced over a certain time. The activities of the different mitochondrial complexes can also be quantified separately. Due to the difficulty in obtaining tissue (like muscle tissue) from paediatric patients we decided to determine mitochondrial respiration in situ in fibroblasts. The method has the advantage that the mitochondria don't have to be isolated and that several tests can be done with a small amount of tissue. It is also a lot easier to get fibroblasts than it is to get paediatric muscle tissue. The cells are made permeable with digitonin so that the mitochondrial complex specific substrates can reach the mitochondria in the cells. We decided to quantify the amount of ATP produced in a certain time by the use of a luciferase-luciferin enzyme system. The method is very sensitive yet very easy to perform. We decided to standardise the method for quantification of in situ mitochondrial respiration due to the variations found in literature concerning this method. The aspects that were standardised included the determination of the optimum concentration digitonin to permeabilise the fibroblasts, optimisation of the luciferase-luciferin enzyme reaction, optimisation of the extraction of ATP from cells, determination of the optimum substrate concentration for the different complexes as well as the linear range for each reaction. In addition, with the use of complex specific inhibitors, it was necessary to determine to what extent the various mitochondrial complexes are specifically responsible for the observed respiration. The in situ method for ATP quantification, after standardisation, was used on fibroblasts from people who seemed to have no defects, for the determination of control values. The method was then tested and evaluated on fibroblasts from two patients with possible inherited metabolic diseases. The one patient was diagnosed with a possible cytochrome c oxidase defect and the other patient with a possible complex I defect. Both the patients' respiration values for the involved complexes were lower than the control respiration values. These results were verified by determining the respiration activities of the individual complexes through classical in vitro analyses. With this, a technique was standardised with which mitochondrial respiration defects can be examined in for example paediatric patients.