Respirometry
Encyclopedia
Respirometry is a general term that encompass a number of techniques for obtaining estimates of the rates of metabolism
of vertebrates, invertebrates, plants, tissues, cells, or microorganisms via an indirect measure of heat production (calorimetry
).
production (VCO2) and oxygen
consumption (VO2) of individual animals, either in a closed or an open-circuit respirometry system. Two measures are typically obtained: standard (SMR) or basal metabolic rate
(BMR) and maximal rate (VO2max). SMR is measured while the animal is at rest (but not asleep) under specific laboratory (temperature, hydration) and subject-specific conditions (e.g., size or allometry), age, reproduction status, post-absorptive to avoid thermic effect of food
) . VO2max is typically determined during aerobic exercise at or near physiological limits . In contrast, field metabolic rate (FMR) refers to the metabolic rate of an unrestrained, active animal in nature . Whole-animal metabolic rates refer to these measures without correction for body mass. If SMR or BMR values are divided by the body mass value for the animal, then the rate is termed mass-specific. It is this mass-specific value that one typically hears in comparisons among species.
into an air-tight container. The air sealed in the container initially contains the same composition and proportions of gases that were present in the room: 20.95% O2
, 0.03% CO2
, water vapor
(the exact amount depends on air temperature, see dew point
), 78% (approximately) N2
, and a variety of trace gases making up the rest (see Earth's atmosphere
). As time passes, the mouse
in the chamber produces CO2 and water vapor, but extracts O2 from the air in proportion to its metabolic demands. Therefore, as long as we know the volume of the system, the difference between the concentrations of O2 and CO2 at the start when we sealed the mouse
into the chamber (the baseline or reference conditions) compared to the amounts present after the mouse
has breathed the air at a later time must be the amounts of CO2/O2 produced/consumed by the mouse
. In a closed system, the environment will eventually become hypoxic
.
, flow rates of about 200 ml/min through 500 ml containers would provide a good balance. At this flow rate, about 40 ml of O2 is brought to the chamber and the entire volume of air in the chamber is exchanged within 5 minutes. For other smaller animals, chamber volumes can be much smaller and flow rates would be adjusted down as well. Note that for warm-blooded
or endothermic animals (birds and mammals), chamber sizes and or flow rates would be selected to accommodate their higher metabolic rates.
consumed, one needs to know the location of the flow meter relative to the animal chamber (if positioned before the chamber, the flow meter is "upstream," if positioned after the chamber, the flow meter is "downstream"), and whether or not reactive gases are present (e.g., CO2, water
, methane
, see inert gas
).
For an open system with upstream flow meter, water (e.g., anhydrous calcium sulfate
) and CO2 (e.g., a carbon dioxide adsorbent like Ascarite II, a registered trademark of the Arthur H. Thomas Co.) removed prior to oxygen
analyzer, a suitable equation is
For an open system with downstream flow meter, water and CO2 removed prior to oxygen
analyzer, a suitable equation is
where
For example, values for BMR of a 20 g mouse (Mus musculus) might be FR = 200 ml/min, and readings of fractional concentration of O2 from an oxygen analyzer are FiO2 = 0.2095, FeO2 = 0.2072. The calculated rate of oxygen consumption is 0.58 ml/min or 35 ml/hour. Assuming an enthalpy
of combustion for O2 of 20.1 joule
s per milliliter, we would then calculate the heat production (and therefore metabolism) for the mouse as 703.5 J/h.
Pumps for air flow
Flow meter and flow controllers
Tubing and chambers
Analyzers
Finally, a computer data acquisition and control system would be a typical addition to complete the system. Instead of a chart recorder
, continuous records of oxygen consumption and or carbon dioxide production are made with the assistance of an analog to digital converter coupled to a computer. Software captures, filters, converts, and displays the signal as appropriate to the experimenter's needs. A variety of companies and individuals service the respirometry community (e.g., Sable Systems
, Qubit Systems, see also Warthog Systems).
Mitochondrial respirometry measures the consumption of oxygen by the mitochondria without involving an entire living animal and is the main tool to study mitochondrial function. Three different types of samples may be subjected to such respirometric studies:
isolated mitochondria (from cell cultures, animals or plats)
permeabilized cells (from cell cultures)
permeabilized fibers or tissues (from animals)
In the latter two cases the cellular membrane is made permeable by the addition of chemicals leaving selectively the mitochondrial membrane intact. Therefore, chemicals that usually would not be able to cross the cell membrane can directly influence the mitochondria. By the permeabilization of the cellular membrane the cell stops to exist a living, defined organism leaving only the mitochondria as still functional structures.
Unlike whole-animal respirometry, mitochondrial respirometry takes place in solution, i.e. the sample is suspended in a medium. Today mitochondrial respirometry is mainly performed with a closed-chamber approach.
. Functional differences between mitochondria from different species are studied by respirometry as an aspect of comparative physiology
.
mitochondrial diseases or diseases with a (suspected) strong link to mitochondria, e.g. diabetes mellitus type 2
, obesity
and cancer
. Other fields of application are e.g. sports science
and the connection between mitochondrial function and aging.
Metabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
of vertebrates, invertebrates, plants, tissues, cells, or microorganisms via an indirect measure of heat production (calorimetry
Calorimetry
Calorimetry is the science of measuring the heat of chemical reactions or physical changes. Calorimetry is performed with a calorimeter. The word calorimetry is derived from the Latin word calor, meaning heat...
).
Whole-animal metabolic rates
The metabolism of an animal is estimated by determining rates of carbon dioxideCarbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
production (VCO2) and oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
consumption (VO2) of individual animals, either in a closed or an open-circuit respirometry system. Two measures are typically obtained: standard (SMR) or basal metabolic rate
Basal metabolic rate
Basal Metabolic Rate , and the closely related resting metabolic rate , is the amount of daily energy expended by humans and other animals at rest. Rest is defined as existing in a neutrally temperate environment while in the post-absorptive state...
(BMR) and maximal rate (VO2max). SMR is measured while the animal is at rest (but not asleep) under specific laboratory (temperature, hydration) and subject-specific conditions (e.g., size or allometry), age, reproduction status, post-absorptive to avoid thermic effect of food
Thermic effect of food
Thermic effect of food , or TEF in shorthand, is the increment in energy expenditure above resting metabolic rate due to the cost of processing food for storage and use. It is one of the components of metabolism along with the resting metabolic rate, and the exercise component...
) . VO2max is typically determined during aerobic exercise at or near physiological limits . In contrast, field metabolic rate (FMR) refers to the metabolic rate of an unrestrained, active animal in nature . Whole-animal metabolic rates refer to these measures without correction for body mass. If SMR or BMR values are divided by the body mass value for the animal, then the rate is termed mass-specific. It is this mass-specific value that one typically hears in comparisons among species.
Closed respirometry
Respirometry depends on a "what goes in must come out" principle . Consider a closed system first. Imagine that we place a mouseMouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
into an air-tight container. The air sealed in the container initially contains the same composition and proportions of gases that were present in the room: 20.95% O2
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
, 0.03% CO2
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
, water vapor
Water vapor
Water vapor or water vapour , also aqueous vapor, is the gas phase of water. It is one state of water within the hydrosphere. Water vapor can be produced from the evaporation or boiling of liquid water or from the sublimation of ice. Under typical atmospheric conditions, water vapor is continuously...
(the exact amount depends on air temperature, see dew point
Dew point
The dew point is the temperature to which a given parcel of humid air must be cooled, at constant barometric pressure, for water vapor to condense into liquid water. The condensed water is called dew when it forms on a solid surface. The dew point is a saturation temperature.The dew point is...
), 78% (approximately) N2
Nitrogen
Nitrogen is a chemical element that has the symbol N, atomic number of 7 and atomic mass 14.00674 u. Elemental nitrogen is a colorless, odorless, tasteless, and mostly inert diatomic gas at standard conditions, constituting 78.08% by volume of Earth's atmosphere...
, and a variety of trace gases making up the rest (see Earth's atmosphere
Earth's atmosphere
The atmosphere of Earth is a layer of gases surrounding the planet Earth that is retained by Earth's gravity. The atmosphere protects life on Earth by absorbing ultraviolet solar radiation, warming the surface through heat retention , and reducing temperature extremes between day and night...
). As time passes, the mouse
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
in the chamber produces CO2 and water vapor, but extracts O2 from the air in proportion to its metabolic demands. Therefore, as long as we know the volume of the system, the difference between the concentrations of O2 and CO2 at the start when we sealed the mouse
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
into the chamber (the baseline or reference conditions) compared to the amounts present after the mouse
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
has breathed the air at a later time must be the amounts of CO2/O2 produced/consumed by the mouse
Mouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
. In a closed system, the environment will eventually become hypoxic
Hypoxia (medical)
Hypoxia, or hypoxiation, is a pathological condition in which the body as a whole or a region of the body is deprived of adequate oxygen supply. Variations in arterial oxygen concentrations can be part of the normal physiology, for example, during strenuous physical exercise...
.
Open respirometry
For an open-system, design constraints include washout characteristics of the animal chamber and sensitivity of the gas analyzers. However, the basic principle remains the same: What goes in must come out. The primary distinction between an open and closed system is that the open system flows air through the chamber (i.e., air is pushed or pulled by pump) at a rate that constantly replenishes the O2 depleted by the animal while removing the CO2 and water vapor produced by the animal. The flow rate must be high enough to ensure that the animal never consumes all of the oxygen present in the chamber while at the same time, the rate must be low enough so that the animal consumes enough O2 for detection. For a 20 g mouseMouse
A mouse is a small mammal belonging to the order of rodents. The best known mouse species is the common house mouse . It is also a popular pet. In some places, certain kinds of field mice are also common. This rodent is eaten by large birds such as hawks and eagles...
, flow rates of about 200 ml/min through 500 ml containers would provide a good balance. At this flow rate, about 40 ml of O2 is brought to the chamber and the entire volume of air in the chamber is exchanged within 5 minutes. For other smaller animals, chamber volumes can be much smaller and flow rates would be adjusted down as well. Note that for warm-blooded
Warm-blooded
The term warm-blooded is a colloquial term to describe animal species which have a relatively higher blood temperature, and maintain thermal homeostasis primarily through internal metabolic processes...
or endothermic animals (birds and mammals), chamber sizes and or flow rates would be selected to accommodate their higher metabolic rates.
Calculations
Calculating rates of VO2 and/or VCO2 requires knowledge of the flow rates into and out of the chamber, plus fractional concentrations of the gas mixtures into and out of the animal chamber. In general, metabolic rates are calculated from steady-state conditions (i.e., animal's metabolic rate is assumed to be constant rates of oxygenOxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
consumed, one needs to know the location of the flow meter relative to the animal chamber (if positioned before the chamber, the flow meter is "upstream," if positioned after the chamber, the flow meter is "downstream"), and whether or not reactive gases are present (e.g., CO2, water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
, methane
Methane
Methane is a chemical compound with the chemical formula . It is the simplest alkane, the principal component of natural gas, and probably the most abundant organic compound on earth. The relative abundance of methane makes it an attractive fuel...
, see inert gas
Inert gas
An inert gas is a non-reactive gas used during chemical synthesis, chemical analysis, or preservation of reactive materials. Inert gases are selected for specific settings for which they are functionally inert since the cost of the gas and the cost of purifying the gas are usually a consideration...
).
For an open system with upstream flow meter, water (e.g., anhydrous calcium sulfate
Calcium sulfate
Calcium sulfate is a common laboratory and industrial chemical. In the form of γ-anhydrite , it is used as a desiccant. It is also used as a coagulant in products like tofu. In the natural state, unrefined calcium sulfate is a translucent, crystalline white rock...
) and CO2 (e.g., a carbon dioxide adsorbent like Ascarite II, a registered trademark of the Arthur H. Thomas Co.) removed prior to oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
analyzer, a suitable equation is
For an open system with downstream flow meter, water and CO2 removed prior to oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
analyzer, a suitable equation is
where
- FR is the flow rate adjusted to STP (see Standard conditions for temperature and pressureStandard conditions for temperature and pressureStandard condition for temperature and pressure are standard sets of conditions for experimental measurements established to allow comparisons to be made between different sets of data...
) - FiO2 is the fractional amount of oxygen present in the incurrent air stream (the baseline or reference), and
- FeO2 is the fractional amount of oxygen present in the excurrent air stream (what the animal has consumed relative to baseline per unit time).
For example, values for BMR of a 20 g mouse (Mus musculus) might be FR = 200 ml/min, and readings of fractional concentration of O2 from an oxygen analyzer are FiO2 = 0.2095, FeO2 = 0.2072. The calculated rate of oxygen consumption is 0.58 ml/min or 35 ml/hour. Assuming an enthalpy
Enthalpy
Enthalpy is a measure of the total energy of a thermodynamic system. It includes the internal energy, which is the energy required to create a system, and the amount of energy required to make room for it by displacing its environment and establishing its volume and pressure.Enthalpy is a...
of combustion for O2 of 20.1 joule
Joule
The joule ; symbol J) is a derived unit of energy or work in the International System of Units. It is equal to the energy expended in applying a force of one newton through a distance of one metre , or in passing an electric current of one ampere through a resistance of one ohm for one second...
s per milliliter, we would then calculate the heat production (and therefore metabolism) for the mouse as 703.5 J/h.
Respirometry equipment
For open flow system, the list of equipment and parts is long compared to the components of a closed system, but the chief advantage of the open system is that it permits continuous recording of metabolic rate. The risk of hypoxia is also much less in an open system.Pumps for air flow
- Vacuum PumpVacuum pumpA vacuum pump is a device that removes gas molecules from a sealed volume in order to leave behind a partial vacuum. The first vacuum pump was invented in 1650 by Otto von Guericke.- Types :Pumps can be broadly categorized according to three techniques:...
: a pump is needed to push (i.e., upstream location) or pull (i.e., downstream location) air into and through the animal chamber and respirometry flow-through system. - Subsample pump: To pull air through the analyzers, a small, stable, reliable pump is used.
Flow meter and flow controllers
- Bubble flow meters: A simple, yet highly accurate way to measure flow rates involves timing movement of bubbles of soap film up glass tubes between marks of known volume. The glass tube is connected at the bottom (for push systems) or at the top (for pull systems) to the air stream. A small rubber pipettePipetteA pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...
bulb attached at the base of the tube acts as both a reservoir and delivery system for the soap bubbles. Operation is simple. First, wet the glass surface along the path bubbles travel (e.g., press the bulb so that copious amounts of soap are pushed up the glass by the air flow) to provide a virtually friction-free surface. Second, pinch the bulb so that one clean bubble is produced. With a stopwatch in hand, record the time required for the bubble to travel between marks on the glass. Note the volume recorded on the upper mark (e.g., 125 = 125 ml), divide the volume by the time required to travel between marks and the result is the flow rate (ml/sec). These instruments can be purchased from a variety of sources, but they may also be constructed from appropriate-sized, glass volumetric pipettesPipetteA pipette is a laboratory tool used to transport a measured volume of liquid.-Use and variations:Pipettes are commonly used in molecular biology, analytical chemistry as well as medical tests...
. - Acrylic flow meters : Under some circumstances of high flow rates we may use simple acrylic flow meters (0 - 2.5 liters/min) to control the flow rates through the metabolic chambers. The meters are located upstream from the metabolic chambers. The flow meters are simple to use but should be calibrated twice daily for use in the respirometry system: once before recording begins (but after the animal has been sealed inside the chamber!!) and again at the end of the recording (before the animal is removed from the chamber). Calibration must be done with a bubble flow meter because the calibration marks on the acrylic meters are only approximate. For proper calibration of flow rates remember that both barometric pressure and temperature of the air streaming through the flow meter (which we assume to be equal to room temperature) must be recorded.
- Mass flow meters: The equations required for calculating rates of oxygen consumption or carbon dioxide production assume that the flow rates into and out of the chambers are known exactly. We use mass flow meters which have the advantage of yielding flow rates independent of temperature and air pressure. Therefore, these flow rates can be considered to be corrected to standard conditions (Standard Temperature Pressure). We only measure and control flow at one location -- downstream from the chamber. Therefore, we must assume that the inflow and outflow rates are identical. However, during construction of the respirometry system, flow rate must be measured at all steps, across all connections, to verify integrity of flow.
- Needle valveNeedle valveA needle valve is a type of valve having a small port and a threaded, needle-shaped plunger. It allows precise regulation of flow, although it is generally only capable of relatively low flow rates.- Construction and operation :...
s: Mass flow meters may be purchased with mass flow controllerMass Flow ControllerA mass flow controller is a device used to measure and control the flow of fluids and gases. A mass flow controller is designed and calibrated to control a specific type of fluid or gas at a particular range of flow rates...
s which permit setting flow rates. These are expensive, however. Respirometry research often will attempt to measure more than one animal at a time, which would necessitate one chamber per animal and thus controlled flow through each chamber. An alternative and more cost-effective method to control flow would be via stainless steel or carbon steel needle valves. Needle valves plus mass flow meters provides a cost-effective means to achieve desired flow rates. The valves cost about $20.
Tubing and chambers
- Tubing and connections : Various kinds of tubing can be use to connect the components of the respirometry system to and from the animal chamber. A variety of kinds of flexible tubing may be used, depending on the characteristics of the system. Acetyl, Bev-A-Line, Kynar, nylon, Tygon tubing and connectors may be used in regions of the system where oxidizing atmospheres are low (e.g., background levels of ozone only); Teflon tubing would be recommended if there is an expectation for appreciable amounts of ozone to be present because it is inert to ozone. Teflon tubes are more costly and lack flexibility.
- Metabolic chambers: Chambers may be glass jars with rubber stoppers for lids; syringe barrels for small animals and insects; or constructed from Plexiglas. Ideally, chambers should be constructed from inert materials; for example, the acrylic plastics can absorb O2 and may be a poor choice for respirometry with very small insects . Chambers need to be constructed in a manner that yields rapid mixing of gases within the chamber. The simplest metabolic chamber for a small vertebrate might be a glass jar with a stopper. The stoppers are fitted with two ports: short extensions of Teflon tubing are provided for line connections. Teflon tube extensions are pushed through the bulkhead and the line connection is finished by attaching a small hose clip to the base of the Teflon tube extension. Additionally, an extension to the inlet port inside the jar should be provided -- this ensures that the animal's expiratory gases are not washed away by the in flow stream. The animal is sealed inside and the rubber stopper is held in place with VelcroVelcroVelcro is the brand name of the first commercially marketed fabric hook-and-loop fastener, invented in 1948 by the Swiss electrical engineer George de Mestral...
straps. If an upstream system is used, any metabolic chamber leak will result in loss of animal air and, therefore, an underestimate of the animal's metabolic rate. When you close an animal inside a metabolic chamber, attention must be paid to the seal. To ensure tight seals before closing the lid, firmly work the stopper into the jar and make sure that it is even. Use 1-2 straps (2 are better) and pull tightly. Acrylic (Plexiglas) chambers will be constructed for some uses, but precise engineering will be needed to ensure proper seating; gaskets will help, and judicious use of tight-fitting clamps will minimize leaks. - Scrubbing tubes: Water before and after the animal chamber must be removed. One arrangement would use a large acrylic column of DrieriteDrieriteDrierite is the brand name and registered trademark of a variety of desiccants made from anhydrous calcium sulfate . Some varieties contain 3% cobalt chloride, a moisture-sensitive color indicator that shows when that material's activity has been depleted. Drierite is manufactured in the USA by the...
(8 mesh (scale)Mesh (scale)Mesh material is often used in determining the particle size distribution of a granular material. For example, a sample from a truckload of peanuts may be placed atop a mesh with 5 mm openings. When the mesh is shaken, small broken pieces and dust pass through the mesh while whole peanuts are...
, i.e., relatively coarse) upstream (before the push pump, before the animal chamber) to dry incurrent airstream and several tubes with smaller mesh (10-20, i.e., relatively fine) Drierite to remove water after the animal chamber. To prepare a scrubbing tube, make sure there is a small amount of cotton at either end of the tube to prevent dust particles from traveling to the analyzers. Use small amounts of cotton, say around 0.005 g, just enough to keep the dust out of the tubing. Large amounts of cotton will block air flow when/if it gets damp. Pour the Drierite into the tube with a funnel, tap the tube on the bench to pack the grains tightly (to increase surface area - air + water rushes through loose Drierite, requiring frequent changes of scrubbers), and cap off with a small amount of cotton. To remove carbon dioxide] before and after the animal chamber, Ascarite II is used (Ascarite II is a registered trademark of the Arthur H. Thomas Co.). Ascarite II contains NaOH, which is caustic (so don't get any on your skin and keep away from water). A scrubbing tube is prepared by placing a small amount of cotton into the tube end, filling one-third of the way with 10-20 mesh Drierite, adding a small amount of cotton, then an additional third of the tube with the Ascarite II, another layer of cotton, followed by more Drierite and capping the tube off with another small amount of cotton. Tap the tube on the bench as each layer is added to pack the grains. Note: Driereite can be used over and over again (after heating in an oven), although indicating Drierite will lose color with repeated drying; Ascarite II is used once and will be considered a hazardous wasteHazardous wasteA hazardous waste is waste that poses substantial or potential threats to public health or the environment. According to the U.S. environmental laws hazardous wastes fall into two major categories: characteristic wastes and listed wastes.Characteristic hazardous wastes are materials that are known...
.
Analyzers
- Carbon dioxideCarbon dioxideCarbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
analyzer: CO2 analyzers typically use infrared-based detection methods to take advantage of the fact that CO2 will absorb infra-red light and re-emit light at slightly longer wavelengths. The panel meter on the analyzer displays over the entire 0.01 - 10% CO2 range and a voltage output proportional to CO2 concentration is also generated for data recording. - OxygenOxygenOxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
analyzer: Oxygen analyzers suitable for respirometry use a variety of oxygen sensorOxygen sensorAn oxygen sensor, or lambda sensor, is an electronic device that measures the proportion of oxygen in the gas or liquid being analyzed. It was developed by the Robert Bosch GmbH company during the late 1960s under the supervision of Dr. Günter Bauman...
s, including galvanicGalvanic cellA Galvanic cell, or Voltaic cell, named after Luigi Galvani, or Alessandro Volta respectively, is an electrochemical cell that derives electrical energy from spontaneous redox reaction taking place within the cell...
("ambient temperature"), paramagnetic, polarographic (ClarkClark electrodeThe Clark electrode is an electrode that measures oxygen on a catalytic platinum surface using the net reaction:- History :Leland Clark had developed the first bubble oxygenator for use in cardiac surgery...
-type electrodes), and zirconiumZirconiumZirconium is a chemical element with the symbol Zr and atomic number 40. The name of zirconium is taken from the mineral zircon. Its atomic mass is 91.224. It is a lustrous, grey-white, strong transition metal that resembles titanium...
("high temperature") sensors. Galvanic O2 analyzers use a fuel cellFuel cellA fuel cell is a device that converts the chemical energy from a fuel into electricity through a chemical reaction with oxygen or another oxidizing agent. Hydrogen is the most common fuel, but hydrocarbons such as natural gas and alcohols like methanol are sometimes used...
containing an acidic electrolyteElectrolyteIn chemistry, an electrolyte is any substance containing free ions that make the substance electrically conductive. The most typical electrolyte is an ionic solution, but molten electrolytes and solid electrolytes are also possible....
, a heavy-metal anodeAnodeAn anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
and a thin gas-permeable membrane. Since the partial pressurePartial pressureIn a mixture of ideal gases, each gas has a partial pressure which is the pressure which the gas would have if it alone occupied the volume. The total pressure of a gas mixture is the sum of the partial pressures of each individual gas in the mixture....
of O2 near the anode is zero, O2 is driven by diffusionDiffusionMolecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
to the anodeAnodeAn anode is an electrode through which electric current flows into a polarized electrical device. Mnemonic: ACID ....
via the membrane at a rate proportional to ambient O2 partial pressure. The fuel cell produces a voltage linearly proportional to the O2 partial pressure at the membrane. As long as cabinet temperature is stable, and provided that air flow across the fuel cell is stable and within range, the response will be 0.01% or better depending on supporting electronics, software, and other considerations.
Finally, a computer data acquisition and control system would be a typical addition to complete the system. Instead of a chart recorder
Chart recorder
A chart recorder is an electromechanical device that records an electrical or mechanical input trend onto a piece of paper . Chart recorders may record several inputs using different color pens and may record onto strip charts or circular charts...
, continuous records of oxygen consumption and or carbon dioxide production are made with the assistance of an analog to digital converter coupled to a computer. Software captures, filters, converts, and displays the signal as appropriate to the experimenter's needs. A variety of companies and individuals service the respirometry community (e.g., Sable Systems
Sable Systems
Sable Systems develops and manufactures equipment for whole animal respirometry and offers courses in respirometry....
, Qubit Systems, see also Warthog Systems).
Mitochondrial metabolic rates
Inside the body oxygen is delivered to cells and in the cells to mitochondria, where it is consumed in the process generating most of the energy required by the organism.Mitochondrial respirometry measures the consumption of oxygen by the mitochondria without involving an entire living animal and is the main tool to study mitochondrial function. Three different types of samples may be subjected to such respirometric studies:
isolated mitochondria (from cell cultures, animals or plats)
permeabilized cells (from cell cultures)
permeabilized fibers or tissues (from animals)
In the latter two cases the cellular membrane is made permeable by the addition of chemicals leaving selectively the mitochondrial membrane intact. Therefore, chemicals that usually would not be able to cross the cell membrane can directly influence the mitochondria. By the permeabilization of the cellular membrane the cell stops to exist a living, defined organism leaving only the mitochondria as still functional structures.
Unlike whole-animal respirometry, mitochondrial respirometry takes place in solution, i.e. the sample is suspended in a medium. Today mitochondrial respirometry is mainly performed with a closed-chamber approach.
Closed-chamber System
The sample suspended in a suitable medium is placed in a hermetically closed metabolic chamber. The mitochondria are brought into defined “states” by the sequential addition of substrates or inhibitors. Since the mitochondria consume oxygen, the oxygen concentration drops. This change of oxygen concentration is recorded by an oxygen sensor in the chamber. From the rate of the oxygen decline (taking into account correction for oxygen diffusion) the respiratory rate of the mitochondria can be computed.Basic research
The functioning of mitochondria is studied in the field of bioenergeticsBioenergetics
Bioenergetics is the subject of a field of biochemistry that concerns energy flow through living systems. This is an active area of biological research that includes the study of thousands of different cellular processes such as cellular respiration and the many other metabolic processes that can...
. Functional differences between mitochondria from different species are studied by respirometry as an aspect of comparative physiology
Comparative physiology
Comparative physiology is a subdiscipline of physiology that studies and exploits the diversity of functional characteristics of various kinds of organisms. It is closely related to evolutionary physiology and environmental physiology. Many universities offer undergraduate courses that cover...
.
Applied research
Mitochondrial respirometry is used to study mitochodrial functionality inmitochondrial diseases or diseases with a (suspected) strong link to mitochondria, e.g. diabetes mellitus type 2
Diabetes mellitus type 2
Diabetes mellitus type 2formerly non-insulin-dependent diabetes mellitus or adult-onset diabetesis a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Diabetes is often initially managed by increasing exercise and...
, obesity
Obesity
Obesity is a medical condition in which excess body fat has accumulated to the extent that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems...
and cancer
Cancer
Cancer , known medically as a malignant neoplasm, is a large group of different diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the...
. Other fields of application are e.g. sports science
Sports science
Sport science is a discipline that studies the application of scientific principles and techniques with the aim of improving sporting performance...
and the connection between mitochondrial function and aging.
Equipment
The usual equipment includes a seal-able metabolic chamber, an oxygen sensor, and devices for data recording, stirring, thermostatisation and a way to introduce chemicals into the chamber. As described above for whole-animal respirometry the choice of materials is very important. Plastic materials are not suitable for the chamber because of their oxygen storage capacity. When plastic materials are unavoidable (e.g. for o-rings, coatings of stirrers, or stoppers) polymers with a very low oxygen permeability (like PVDF as opposed to e.g. PTFE) may be used. Remaining oxygen diffusion into or out of the chamber materials can be handled by correcting the measured oxygen fluxes for the instrumental oxygen background flux. The entire instrument comprising the mentioned components is often called an oxygraph. The companies providing equipment for whole-animal rspirometry mentioned above are usually not involved in mitochondrial respiromety. The community is serviced at widely varying levels of price and sophistication by companies like Oroboros Instruments, Hansatech, Respirometer Systems & Applications, YSI Life Sciences or Strathkelvin Instruments .See also
- Basal metabolic rateBasal metabolic rateBasal Metabolic Rate , and the closely related resting metabolic rate , is the amount of daily energy expended by humans and other animals at rest. Rest is defined as existing in a neutrally temperate environment while in the post-absorptive state...
- CalorimetryCalorimetryCalorimetry is the science of measuring the heat of chemical reactions or physical changes. Calorimetry is performed with a calorimeter. The word calorimetry is derived from the Latin word calor, meaning heat...
- MetabolismMetabolismMetabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
- VO2max
- RespirometerRespirometerA respirometer is a device used to measure the rate of respiration of a living organism by measuring its rate of exchange of oxygen and/or carbon dioxide. They allow investigation into how factors such as age, chemicals or the effect of light affect the rate of respiration...