Respirometry

Estimation of metabolic rates by measuring heat production
Respirometry is a general term that encompasses 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).

The metabolism of an animal is estimated by determining rates of carbon dioxide 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.

Respirometry depends on a "what goes in must come out" principle. Consider a closed system first. Imagine that we place a mouse 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, 0.04% CO2, water vapor (the exact amount depends on air temperature, see dew point), 78% (approximately) N2, 0.93% argon 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. Nitrogen and argon are inert gasses and therefore their fractional amounts are unchanged by the respiration of the mouse. In a closed system, the environment will eventually become hypoxic.

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 volumetric 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 mouse, 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.

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 plants); permeabilized cells (from cell cultures); and 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 as 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.

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. Functional differences between mitochondria from different species are studied by respirometry as an aspect of comparative physiology.

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 .