Detection of mitochondrial dysfunction by blood mononuclear cells analysis

Since mitochondria play diverse crucial roles within the cell, mitochondrial dysfunction can have disastrous consequences. High resolution respirometry (HRR) is a new tool that allows the quantification of mitochondrial oxidative phosphorylation and electron transfer capacities. This tool allows an analysis of mitochondrial bioenergetics and the main substrate pathway of the cells that are of interest. In human, platelets and peripheral blood mononuclear cells have been used as biomarkers in diverse pathological models, as diabetes, atherosclerosis, cancer and neurodegenerative diseases.
In the horse, HRR studies have been conducted on skeletal muscles samples. Using this technique, a so-called “mitochondrial syndrome” has been reported in equine atypical myopathy, an environmental intoxication, which is characterized by a severe decrease of mitochondrial respiratory capacity in affected individuals. However, the muscle biopsy procedure is difficult to use in equine clinical routine. Therefore, the aim of this study was to test whether mitochondrial function could be assessed on equine blood mononuclear cells with respirometry. Whole blood was collected in 9 ml EDTA tubes in six horses. Cells of interest (PBMC) were isolated with a density-separation medium (Lymphoprep™) following a standardized procedure. Electron paramagnetic resonance as well as HRR analysis were performed within the following 6 hours. With this isolation technique more than 1x106 PBMC/ml of whole blood were obtained. Respirometric analysis with PBMC taken from the same horses indicated a lack of reproducibility. This finding seems to be primarily due to a too long timeframe (i.e. ±Xh) between cell isolation and respirometric analysis. Additional studies indicated that when respirometry was performed within the first 2 hours after cell isolation, standard variation between samples was significantly reduced. Up to now a correlation between skeletal muscle fiber and PBMC mitochondrial bioenergetics in the equine species is not possible to be established.
A standardization of the procedure is vital for reproducibility between laboratories and comparison between healthy and ill horses. The next steps are to compare the oxygraphic measures between equine PBMC and muscle biopsies, as well as the comparison of mitochondrial function between healthy and horses in extreme metabolic conditions (e.g atypical myopathy).