Authors
Matthew D Hirschey and Eric Verdin
Summary
Organisms store excess energy as fatty acids and triglycerides. Under fasting conditions, fatty acids are oxidized in the mitochondria in a multi-step pathway called beta-oxidation1. Beta-oxidation (or fatty acid oxidation) begins by importing long-chain fatty acids into the mitochondria, followed by a four-step reaction. In the first step, acyl-CoA dehydrogenase dehydrogenates the long-chain acyl-CoA. In the second step, enoyl-CoA hydratase hydrates the intermediate forming a hydroxy-acyl-CoA. In the third step, hydroxy-acyl-CoA dehydrogenase oxidizes the intermediate forming a keto-group. In the fourth step, thiolase cleaves acetyl-CoA and adds CoA to the new substrate, generating an acyl-CoA shortened by two carbons. This cycle is repeated until the entire chain is oxidized into acetyl-CoA. Mitochondrial fatty acid oxidation can be measured at the level of individual enzymes or by measuring flux through the pathway2. Enzymatic activity is measured in cell culture in vitro or in tissue homogenate ex vivo, whereas pathway flux can be measured in vitro, ex vivo, or indirectly in vivo3. Here, we describe detailed methods adapted from previous studies4-7 using common supplies and reagents for preparing hepatic, cardiac, skeletal muscle and adipose tissue homogenates to measure fatty acid oxidation flux ex vivo (Figure 1).Further details
The protocol was published on Protocol Exchange on 15 April 2010. To see the entire protocol, click on the source link.Advertisement
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