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The best approach: Homogenization or manual permeabilization of human skeletal muscle fibers for respirometry?

Author:
Larsen, Steen, Kraunsøe, Regitze, Gram, Martin, Gnaiger, Erich, Helge, Jørn W., Dela, Flemming
Source:
Analytical biochemistry 2014 v.446 pp. 64-68
ISSN:
0003-2697
Subject:
biopsy, cytochrome c, homogenization, humans, mitochondria, mitochondrial membrane, muscles, myotubes, oxygen consumption, permeability, skeletal muscle
Abstract:
The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by the mitochondria in human skeletal muscle are used in many studies. There are several advantages of studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated whether the PBI shredder is useful in preparing a muscle homogenate for measurements of mitochondrial respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for preparing human skeletal muscle biopsies for respirometry.
Agid:
5983817