Razak 2018 RUMeS
|Razak SHA, Hassan H, Hakimi NH, Rani NA, Ibrahim NF, Ngah WZW, Makpol S, Damanhuri MHA, Karim NA, Tan JK (2018) Isolation of mitochondria and measurement of oxygen consumption rate by high-resolution respirometry in rat hippocampus. RUMeS 6:11-18.|
Abstract: Hippocampus plays an important role in memory and learning. Dysfunction of mitochondrial bioenergetics has been implicated in neurodegenerative disorders such as Alzheimer disease. Oxygen consumption rate (OCR) is a useful indicator of mitochondrial function. Mitochondria isolated for OCR measurement need to have well intact function. This study aimed to describe the measurement of OCR by high resolution respirometry (HRR) in mitochondria isolated from rat hippocampus.
Protocol of mitochondrial isolation was based on sucrose step density gradient centrifugation method as adapted from previous study. OCR of isolated mitochondria was measured using OROBOROS HRR Oxygraph-2k according to substrate-uncoupler-inhibitor titration (SUIT) protocol.
Our results showed that OCR increased following the addition of complex I-linked substrates (glutamate, pyruvate, and malate), coupling substrate (adenosine dinucleotide) and complex II-linked substrate (succinate). OCR decreased following the addition of ATP synthase inhibitor (oligomycin), while maximal respiration was achieved following the addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) uncoupler. Residual oxygen consumption (ROX) was obtained following the addition of complex I inhibitor (rotenone) and complex III inhibitor (antimycin A).
Taken together, our findings suggested that mitochondria isolated from this protocol can be used for measurement of OCR using HRR. Measurement of OCR offers meaningful information regarding the mitochondrial bioenergetics states of a sample.
Labels: MiParea: Respiration
Organism: Rat Tissue;cell: Nervous system Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k