Osiki 2016 FASEB J
|Osiki PO, Ojuka E, Marmaan G (2016) An investigation of the suitability of using octanoylcarnitine together with malate as a substrate combination for assessing Beta-oxidation using High Resolution Respirometry. FASEB J 30: 1015.12 .|
Abstract: Background Beta-oxidation is often measured using respirometry with octanoylcarnitine + malate as substrates in cells. Malate is necessary to ensure continuous oxidation of octanoylcarnitine. However, since malate is metabolized in the TCA cycle, it is not clear if its inclusion as a co-substrate allows for a valid assessment of beta-oxidation when TCA cycle function is compromised.
Aim To investigate the validity of beta-oxidation assessment using octanoylcarnitine + malate as a substrate combination in skeletal muscle when mitochondrial (mt) aconitase is inhibited.
Methods Soleus muscle fibres (1.5–2mg) from healthy male Wistar rats were permeabilized with saponin and incubated for 45 minutes with 1mM oxalomalic acid (aconitase inhibitor) or 1mM 2-mercaptoacetate, an inhibitor of MCAD – the rate-limiting enzyme of octanoylcarnitine oxidation. Respiration at Leak, Oxphos and ET-pathway states were measured using an Oroboros oxygraph. Citrate and 2-oxoglutarate in the respiratory media were measured using CG-MS. Activities of aconitase and MCAD were determined spectrophotometrically.
Results Oxalomalic acid (1mM) and 1mM of 2-mercaptoacetate caused 24% and 58% inhibition of acnonitase and MCAD, respectively. Oxygen flux at Oxphos (0.5 ± 0.3 pmol.S−1.mg−1) and ET-pathway (0.6 ± 0.2 pmol.S−1.mg−1) decreased in 2-mercaptoacetate-treated samples by 62.5% and 60%, respectively, but were unchanged with oxalomalic acid treatment. Respiration at leak state was similar for all treatments. Citrate level in the medium increased by 2-fold at Oxphos state after 30 minutes.
Conclusion Octanoylcarnitine + malate allows for a valid assessment of beta-oxidation capacity using respirometry under conditions where mt-aconitase has been inhibited.
Support or Funding Information Support: 1. The research unit for Exercise Science & Sports Medicine at the University of Cape Town 2. The National Research Foundation (NRF), South Africa, for funding the research.
Footnotes This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Labels: MiParea: Respiration, Instruments;methods
Organism: Rat Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Coupling state: LEAK, OXPHOS, ET Pathway: F HRR: Oxygraph-2k