The best approach: homogenization or manual permeabilization of human skeletal muscle fibers for respirometry?
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The best approach : homogenization or manual permeabilization of human skeletal muscle fibers for respirometry? / Larsen, Steen; Kraunsøe, Regitze; Gram, Martin; Gnaiger, Erich; Helge, Jørn Wulff; Dela, Flemming.
I: Analytical Biochemistry, Bind 446, Nr. 1, 01.02.2014, s. 64-8.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - The best approach
T2 - homogenization or manual permeabilization of human skeletal muscle fibers for respirometry?
AU - Larsen, Steen
AU - Kraunsøe, Regitze
AU - Gram, Martin
AU - Gnaiger, Erich
AU - Helge, Jørn Wulff
AU - Dela, Flemming
N1 - Copyright © 2013 Elsevier Inc. All rights reserved.
PY - 2014/2/1
Y1 - 2014/2/1
N2 - 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.
AB - 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.
U2 - 10.1016/j.ab.2013.10.023
DO - 10.1016/j.ab.2013.10.023
M3 - Journal article
C2 - 24161612
VL - 446
SP - 64
EP - 68
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -
ID: 106713865