Respirometry

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Respirometry

Description

Respirometry is the quantitative measurement of respiration. Respiration is therefore a combustion, a very slow one to be precise (Lavoisier and Laplace 1783). Thus the basic idea of using calorimetry to explore the sources and dynamics of heat changes was present in the origins of bioenergetics (Gnaiger 1983). Respirometry provides an indirect calorimetric approach to the measurement of metabolic heat changes, by measuring oxygen uptake (and carbon dioxide production and nitrogen excretion in the form of ammonia, urea or uric acid) and converting the oxygen consumed into an enthalpy change, using the oxycaloric equivalent. Liebig (1842) showed that the substrate of oxidative respiration was protein, carbohydrates, and fat. The sum of these chemical changes of materials under the influence of living cells is known as metabolism (Lusk 1928). The amount (volume STP) of carbon dioxide expired to the amount (volume STP) of oxygen inspired simultaneously is the respiratory quotient, which is 1.0 for the combustion of carbohydrate, but less for lipid and protein. Voit (1901) summarized early respirometric studies carried out by the Munich school on patients and healthy controls, concluding that the metabolism in the body was not proportional to the combustibility of the substances outside the body, but that protein, which burns with difficulty outside, metabolizes with the greatest ease, then carbohydrates, while fats, which readily burns outside, is the most difficultly combustible in the organism. Extending these conclusions on the sources of metabolic heat changes, the corresponding dynamics or respiratory control was summarized (Lusk 1928): The absorption of oxygen does not cause metabolism, but rather the amount of the metabolism determines the amount of oxygen to be absorbed. .. metabolism regulates the respiration.


MitoPedia concepts: MiP concept 


MitoPedia methods: Respirometry 

History

  • Liebig Justus von (1842) Die organische Chemie in ihrer Anwendung auf Physiologie und Pathologie. Braunschweig. »Open Access

External and internal respiration

The brief historical summary on respirometry and respiration in the description (above) illustrates that early concepts on respiration focussed on the mechanism of respiration in living systems. This integrated systems view is different from the notion of respiration separating the external mode of respiration (inhaling and exhaling gas through the lungs, gills or body surface) from the internal mode of cell respiration related to metabolism. The integrated concept of respirometry and respiration thus unifies respirometry applied to ecological systems, whole organims, organs and tissues, living cells, and mitochondria.


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" [1].

Methods and conclusions: The beginning of respiratory physiology and bioenergetics

"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" [1].

1983: Instrumental background 200 years later

"Due to the unfavorable volume-to-surface ratio in animal chambers with a volume less than 0.5 to 1 cm3, increasing attention has to be paid to problems of oxygen diffusion and bacterial growth" [2].

Instrumental background in the O2k

Automatic determination and correction for instrumental background oxygen flux [3].


References
  1. 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.
  2. 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.
  3. MiPNet14.06 Instrumental O2 background.


Keilin 1929: Respiratory systems - respiratory control

'..the O2 uptake of intact cells represents the global result of the activity of several respiratory systems. .. The activity of this system depends therefore on a certain tension of oxygen, on the activity of oxidase, on the presence and distribution of suitable carriers (cytochrome and possibly other as yet unknown substances), on the activity of dehydrases, and on the presence of suitable molecules (metabolites) for activation. It is easy now to conceive conditions under which any one of these constituents may become a limiting factor in the respiratory process.' (Keilin 1929 Proc R Soc London Ser B).


Warburg apparatus

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 proportionally 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 min 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 the recent literature when no clear distinction is made between the Crabtree effect [4] and the Warburg effect.
References
  1. "The Nobel Prize in Physiology or Medicine 1931". Nobelprize.org. 27 Dec 2011 http://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."


Links

  • Respiratory control
  1. Property:Respiration and regulation
  2. Property:Coupling states
  3. Property:Substrate states
  • Instrumental platform
  1. Property:Instrument and method
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