Abstract
Methods Muscle biopsies were obtained from critically ill patients with severe ICUAW (defined as MRC score < 24 on two separate tests) and from otherwise healthy controls undergoing elective hip replacement. 50-100mg of the native sample was homogenized by a technique which perserves mitochondria in their cytosolic context [3]. Oxygen consumption was measured by high-resolution respirometry (Oxygraph-2Km Oroboros) under two occasions: 1. homogenate enriched with substrates was sequentially treated with an ATPase inhibitor (oligomycine), an uncoupler (FCCP) and KCN. This allowed us to measure oxidative phosphorylation, respiratory chain capacity, proton leak through inner mitochondrial membrane and non-mitochondrial O2 consumption. 2. skeletal muscle homogenate enriched with abundant ADP was treated with sequential addition of substrates and inhibitors of complexes I, II, III, and IV, respectively. Integrity of outer membrane was assessed by measuring the response to addition of cytochrome c. Oxygen consumption rate was normalized to citrate-synthase activity and total protein content.
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