Wollenman 2017 PLOS ONE

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Wollenman LC, Vander Ploeg MR, Miller ML, Zhang Y, Bazil JN (2017) The effect of respiration buffer composition on mitochondrial metabolism and function. PLOS ONE 12:e0187523.

» PMID: 29091971 Open Access »O2k-brief

Wollenman LC, Vander Ploeg MR, Miller ML, Zhang Yizhu, Bazil JN (2017) PLOS ONE

Abstract: Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha-ketoglutarate exchanger are partially inhibited by chloride. Therefore, when the goal is to maximize ADP-stimulated respiration, buffers containing K-lactobionate or K-gluconate are superior choices compared to the standard KCl-based buffers.


Bioblast editor: Kandolf G O2k-Network Lab: US TN Nashville Wasserman DH, US MI East Lansing Bazil J

Respiration media

  • B1, KCl-based: 130 mM potassium chloride, 5 mM dibasic potassium phosphate, 1 mM magnesium chloride, 20 mM MOPS, 1 mM EGTA, 0.1% (w/v) BSA at pH 7.1 at 37 °C
  • MiR05, B2: 110 mM sucrose, 60 mM potassium lactobionate, 20 mM taurine, 10 mM monobasic potassium phosphate, 3 mM magnesium chloride, 20 mM HEPES, 1 mM EGTA, and 0.1% (w/v) BSA at pH 7.1 at 37 °C.
  • B3: 130 mM potassium gluconate, 5 mM dibasic potassium phosphate, 1 mM magnesium chloride, 20 mM MOPS, 1 mM EGTA, and 0.1% (w/v) BSA at pH 7.1 at 37 °C.
  • B4: 110 mM sucrose, 60 mM potassium gluconate, 20 mM taurine, 10 mM monobasic potassium phosphate, 3 mM magnesium chloride, 20 mM HEPES, and 0.1% (w/v) BSA at pH 7.1 at 37 °C.

Substrate combinations


Selected quotations

  • In general, buffer B1 led to the lowest respiration rates for any substrate combination.
  • Relative to the other substrate combination profiles, the G/M, αKG/M, and αKG/G (for only B1) combination respiratory profiles showed a substrate- and buffer-dependent delay ranging from 20 to 50 sec until the maximum ADP-stimulatory rate was achieved. This delay was not present when low concentrations of free calcium was present (see S1 Fig).
  • the buffers had very similar leak state respiratory rates with few exceptions.
  • ADP-stimulated respiration rates .. were generally higher across all substrate combinations for the buffers with lower chloride concentrations (Fig 2C).
  • The FCCP uncoupled respiration rates for certain substrate combinations also showed higher rates for the buffers containing low chloride (Fig 2D).
  • The most likely targets of chloride inhibition are the adenine nucleotide translocase and substrate transporters involved with succinate, glutamate, and palmitoylcarnitine catabolism.
  • Overall, the P/M, P/M/S, and S/Rot uncoupled respiration rates were typically higher than the maximal coupled respiration rates (Fig 2F). For PC/M, the maximal uncoupled rate was roughly equal to the maximal coupled rate.
  • Surprisingly, the uncoupled rates were much lower than the coupled rates for the G/M, αKG/M, and αKG/G substrate combinations. The exact reason for this is unknown but may be related to FCCP interfering with substrate transport and/or inhibiting the electron transport system.
  • Surprisingly, the uncoupled rates were much lower than the coupled rates for the G/M, αKG/M, and αKG/G substrate combinations. The exact reason for this is unknown but may be related to FCCP interfering with substrate transport and/or inhibiting the electron transport system.
  • The lower levels {of Ca2+ in the absence of EGTA} found in B2 {compared to B1} are due to lactobionate's calcium chelating properties [21] and can be beneficial when designing mitochondrial calcium-loading experiments. Unfortunately, the formation calcium-lactobionate precipitation can prevent loading of moderate to high calcium concentrations and must be considered when testing mitochondrial function at different calcium loads.
  • .. calcium-gluconate precipitation will be an even worse problem for calcium loading experiments, as its solubility is much lower than the solubility of calcium-lactobionate [21].
  • In conclusion, we show that traditional KCl-based respiratory buffers contain inhibitory levels of chloride that can lower both ADP-stimulated and FCCP-stimulated respiration.

Comments

  • Respiration in units [nmol O2/mg/min]
» EG: to convert to SI units [pmol·s-1·mL-1], multiply values in the above unit by 16.67 (Gnaiger et al 2019)
  • "We calculated that the average oxygen consumption rate due to the electrodes in each chamber was 0.52 +/- 4.20 pmol O2/s/mL."
» EG: The correct value should be about 2.5 pmol·s-1·mL-1 (Gnaiger 2008).
  • Palmitoylcarnitine&malate, PalM, at 5 mM Pal and 2 mM
» EG: At high malate concentration of 2 mM, activation of flux may override F-pathway capacity in the malate anaplerotic pathway control state (Gnaiger 2014). A lower malate concentration (0.1 mM) is recommended (Doerrier et al 2018).
  • "In general, the data reveal that buffer B2 leads to an increase of 30%, 56%, and 18% in the leak state respiration rate, ADP-stimulated rate, and computed the RCR, respectively, as shown in Fig 4B." (liver mitochondria)
» EG: For statistical reasons, it is suggested to use biochemical coupling efficiency instead of RCR (Gnaiger 2014).
  • "The second buffer (B2) is a hybrid buffer consisting of 110 mM sucrose, 60 mM potassium lactobionate, 20 mM taurine, 10 mM monobasic potassium phosphate, 3 mM magnesium chloride, 20 mM HEPES, 1 mM EGTA, and 0.1% (w/v) BSA at pH 7.1 at 37°C. This buffer is from the MiR05 recipe."
» LC: The concentration of EGTA ir MiR05 is 0.5 mM (Gnaiger et al. 2000).
References
  1. Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«
  2. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley & Sons, Inc, Hoboken, NJ:327-52. - »Bioblast link«
  3. Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp. - »Bioblast link«
  4. Gnaiger E, Aasander Frostner E, Abdul Karim N, Abumrad NA et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001.v3. - »Bioblast link«
  5. Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Life in the Cold (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York:431-42. - »Bioblast link«


Labels: MiParea: Respiration 


Organism: Guinea pig  Tissue;cell: Heart, Liver  Preparation: Isolated mitochondria  Enzyme: Adenine nucleotide translocase, Inner mt-membrane transporter  Regulation: Ion;substrate transport  Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, NS  HRR: Oxygraph-2k 

2018-01 


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