Respirometry

High-Resolution Respirometry

  • High-resolution respirometry (HRR) is based on the OROBOROS Oxygraph-2k, combining chamber design, application of oxygen-tight materials, electrochemical sensors and electronics, Peltier-temperature control and software features (DatLab) to obtain a unique level of quantitative resolution of oxygen concentration and oxygen flux, with a closed-chamber or open-chamber mode of operation (TIP2k). Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background oxygen flux (systemic flux compensation) provide the experimental basis for high accuracy of quantitative results and quality control in HRR. - @Bioblast HRR



1783: Earliest report on oxygen backdiffusion and instrumental background

 

"Now in putting the animal under the bell jar and in withdrawing it, we observed that the outisde air penetrated slightly into the interior, along the body of the animal, although immersed partly in the mercury. The mercury does not adhere to the surface of the hair and the skin closely enough to prevent all contact between the outside air and that insiede the bell jar; thus the gas appears less diminshed by the respiration than in fact it is. ... One could fear, moreover, that a part of fixed air that was combined might be due to the atmospheric air. To reassure ourselves on this point, we repeated the same experiment with no guinea pig under the jar. In this case there was no increase in weight of the flasks. That of the second flaks decreased by 4 or 5 grains, doubtless from the evaporation of the water of ist alkali solution."

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later: 

Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166. 

Instrumental background in the O2k

 

Methods and conclusions

 

"We have already said, and we cannot stress this fact too much, that it is less the result of our experiments than the method we have used that we offer to scientists, inviting them, if this methods seems to offer some advantage, to check these experiments which we ourselves propose to repeat with the greatest care."

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.



The Warburg apparatus



* Warburg apparatus @Bioblast

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices. The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his "discovery of the nature and mode of action of the respiratory enzyme" [1].  The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

   The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of 'titrations' can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.

   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C. The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C. Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations. 

   Between 1908 and 1914, Warburg was affiliated with the Naples Marine Biological Station, also known as the Stazione Zoologica, in Naples, Italy.

   Warburg's concept of reduced respiration as a cause of cancer was controversial at the time of the original publication, and remains controversial up-to-date in discussions of the Warburg effect.  In addition, some confusion is caused in the recent literature when no clear distinction is made between the Crabtree effect [4] and the Warburg effect.

  1. "The Nobel Prize in Physiology or Medicine 1931". Nobelprize.org. 27 Dec 2011 www.nobelprize.org/nobel_prizes/medicine/laureates/1931/
  2. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.  
  3. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg's contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  4. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. - "At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells."



The Clark Oxygen Sensor

 

Dr. Leland Clark - inventor of the "Clark Oxygen Sensor" (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications  - Obituary; Review

 

Convenience and simplicity

 

'The convenience and simplicity of the polarographic 'oxygen electrode' technicque for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the invetigator's ingenuity and specific requirements of the system under investigation.'

Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.

 

More precise information

"However, one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.”

Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.


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