Koopman 2016 Nat Protoc

From Bioblast
Publications in the MiPMap
Koopman M, Michels H, Dancy BM, Kamble R, Mouchiroud L, Auwerx J, Nollen EA, Houtkooper RH (2016) A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans. Nat Protoc 11:1798-816.

Β» PMID: 27583642 Open Access

Koopman M, Michels H, Dancy BM, Kamble R, Mouchiroud L, Auwerx J, Nollen EA, Houtkooper RH (2016) Nat Protoc

Abstract: Mitochondrial dysfunction is at the core of many diseases, ranging from inherited metabolic diseases to common conditions that are associated with ageing. While associations between ageing and mitochondrial function have been identified using mammalian models, much of the mechanistic insight has emerged from C. elegans. Mitochondrial respiration is recognized as an indicator of mitochondrial health. Seahorse XF96 respirometers are the state-of-the-art platform to assess respiration in cells, and we adapted the technique for applications involving C. elegans. Here, we provide a detailed protocol to optimise and measure respiration in C. elegans with the XF96 respirometer, including the interpretation of parameters and results. The protocol takes ~2 days to complete, excluding time spent culturing C. elegans, and includes (i) the preparation of C. elegans samples, (ii) selection and loading of compounds to be injected, (iii) preparing and executing a run with the XF96 respirometer, and (iv) post-experimental data-analysis, including normalization. In addition, we compare our XF96 application with other existing techniques, including the 8-well Seahorse XFp. The main benefits of the XF96 include the limited number of worms required and high-throughput capacity due to 96-well format.

β€’ Bioblast editor: Gnaiger E

Koopman 2016 Nat Protoc CORRECTION.png

Correction: FADH2 and Complex II

Ambiguity alert.png
FADH2 is shown as the substrate feeding electrons into Complex II (CII). This is wrong and requires correction - for details see Gnaiger (2024).
Gnaiger E (2024) Complex II ambiguities ― FADH2 in the electron transfer system. J Biol Chem 300:105470. https://doi.org/10.1016/j.jbc.2023.105470 - Β»Bioblast linkΒ«

Hydrogen ion ambiguities in the electron transfer system

Communicated by Gnaiger E (2023-10-08) last update 2023-11-10
Electron (e-) transfer linked to hydrogen ion (hydron; H+) transfer is a fundamental concept in the field of bioenergetics, critical for understanding redox-coupled energy transformations.
Ambiguity alert H+.png
However, the current literature contains inconsistencies regarding H+ formation on the negative side of bioenergetic membranes, such as the matrix side of the mitochondrial inner membrane, when NADH is oxidized during oxidative phosphorylation (OXPHOS). Ambiguities arise when examining the oxidation of NADH by respiratory Complex I or succinate by Complex II.
Ambiguity alert e-.png
Oxidation of NADH or succinate involves a two-electron transfer of 2{H++e-} to FMN or FAD, respectively. Figures indicating a single electron e- transferred from NADH or succinate lack accuracy.
Ambiguity alert NAD.png
The oxidized NAD+ is distinguished from NAD indicating nicotinamide adenine dinucleotide independent of oxidation state.
NADH + H+ β†’ NAD+ +2{H++e-} is the oxidation half-reaction in this H+-linked electron transfer represented as 2{H++e-} (Gnaiger 2023). Putative H+ formation shown as NADH β†’ NAD+ + H+ conflicts with chemiosmotic coupling stoichiometries between H+ translocation across the coupling membrane and electron transfer to oxygen. Ensuring clarity in this complex field is imperative to tackle the apparent ambiguity crisis and prevent confusion, particularly in light of the increasing number of interdisciplinary publications on bioenergetics concerning diagnostic and clinical applications of OXPHOS analysis.

Labels: MiParea: Respiration 

Organism: Caenorhabditis elegans, Nematodes 

Preparation: Intact organism 

HRR: Oxygraph-2k 

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