Flow-through respirometry applied to chamber systems: Pros and cons, hints and tips

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Abstract

Flow-through respirometry is a powerful, accurate methodology for metabolic measurement that is applicable to organisms spanning a body mass range of many orders of magnitude. Concentrating on flow-through respirometry that utilizes a chamber to contain the experimental animals, we describe the most common flow measurement and control methodologies (push, pull and stop-flow) and their associated advantages and disadvantages. Objective methods for calculating air flow rates through the chamber, based on the body mass and taxon of the experimental organism, are presented. Techniques for removing the effect of water vapor dilution, including the direct measurement of water vapor pressure and mathematical compensation for its presence, are described and evaluated, as are issues surrounding the analysis of one or both of the respiratory gases (oxygen and carbon dioxide), and issues related to the mathematical correction of wash-out phenomena (response correction). Two important biomedical applications of flow-through respirometry (metabolic phenotyping and room calorimetry) are discussed in detail, and we conclude with a list of suggestions aimed primarily at investigators starting out in applying flow-through respirometry.

Introduction

Respirometry is a versatile, powerful tool for scientific discovery in animal biology. Because the metabolic rate of an animal is affected by many parameters, both external (e.g. temperature and gas concentrations) and internal (e.g. hormonal state and activity level), it affords a unique insight into an animal's energy balance and overall level of physiological integrity. This paper discusses and evaluates methods of measuring the metabolism of animals in real time using respirometry. It is a necessarily idiosyncratic synthesis of the field; in particular we concentrate on aerial, chamber, flow-through respirometric techniques.

Making the metabolic measurement is just the beginning; such measurements also need to be interpreted from a position of knowledge. Take the example of a small mammal such as a mouse measured by biomedical obesity researchers world-wide at the common animal-facility temperature of 23 °C: its metabolic rate is a complex function of not only its hormonal state and activity level, as alluded to above, but its reaction to thermal stress (23 °C is well below the lower limits of a mouse's thermal neutral zone); the quality and distribution of its pelage (ob/ob mice cannot groom themselves effectively and develop dorsal bald patches which act as thermal windows), and of its ability to induce piloerection of such pelage as it owns; its responses to peripheral vasomotor constriction; the depth and distribution of its lipid deposits, which in the case of some mouse strains such as ob/ob are impressively large and will act as thermal insulators; the distribution and degree of activation of thermogenic brown adipose tissue; the integrity of its lungs and cardiovascular system; its body mass and resulting surface area to volume ratio and other allometric scaling considerations; and so on.

However, while a reminder of the complexity of the phenomena underlying these measurements is in order, the emphasis of the present paper is on understanding the flow-through method and its strengths and limitations rather than on interpreting the results of such measurements. The two primary modes of flow-through respirometry are focused on: push mode and pull mode, including examination of some new flow-through respirometry developments. First, however, we examine some issues common to both respirometric modes.

Section snippets

Compensating for water vapor and CO2 dilution, and barometric pressure

Irrespective of the mode of flow-through respirometry, a central problem that all researchers must solve is the interaction between gas species in the air streams to be analyzed. This is especially critical in the case of oxygen (O2; see also Withers, 2001 for an excellent discussion of this topic). If it is diluted by another gas species, the effect of that dilution is indistinguishable from the removal of O2 from the air stream. O2 is present in very high background concentrations — typically

Stop-flow respirometry

Before proceeding to a detailed discussion of push- and pull-mode respirometry, stop-flow respirometry is briefly broached. Stop-flow respirometry is a peculiar form of flow-through respirometry, primarily used for obtaining the metabolic rates and/or RER of animals too small for the available gas analyser to measure directly using flow-through respirometry. Stop-flow respirometry is simple in principle. Briefly, the chamber is flushed, typically with ambient air, and then sealed for a period

Push mode vs. pull mode respirometry

Flow-through respirometry exists in a few distinct forms, but all of them share a common methodological key; the real-time effect of an organism on the gas concentrations surrounding it. Of the flow-through respirometry methodologies, the two most important are push and pull mode respirometry. In push mode respirometry, a gas stream (usually ambient air) is pushed at a defined flow rate past an animal enclosed in a chamber while in pull mode respirometry the gas stream is instead generated

Response (‘instantaneous’) correction

Because the study organism's expired air mixes with incurrent air in the respirometry chamber and then flows to the gas analyser(s), changes in V˙O2 and V˙CO2 are not instantly reflected in the sample air stream. The delay can be apportioned into two parts. First, the lag time, which is the time required for a change in gas concentrations in the respirometry chamber to reach the analyser (note that this does not account for the response time of the analyser and is a function of the sampling

Baselining

‘Baselining’ means measuring the incurrent gas concentrations of the flow-through respirometry system. This is especially critical in the case of O2 analysers, which are tasked with measuring tiny changes from a huge offset of 20.94%. Because the respirometry equations require knowledge of the concentration difference between incurrent and excurrent air streams, it is imperative to measure incurrent as well as excurrent gas concentrations regularly. This can be done by manual switching of the

Choosing flow rates

The selection of appropriate flow rates for a given subject animal is more straightforward than is often realized. When employing standard equations, i.e. without response correction, to calculate V˙O2 and V˙CO2, the selection is based on the weighting of three main factors: the body mass and taxon of the animal, the quality of equipment being used, and the desired temporal resolution (the time taken for respiratory gas plateaus to be reached once a change in respiratory gas exchange rate has

Production and validation of flow rates

Measurement of flow rate is the single most significant determinant of the accuracy of a typical flow-through respirometry system. In most cases a mass flow control valve provides a flow rate through the chamber at a value referred to ‘standard temperature and pressure’ (STP), though it should be noted that the STP condition is re-defined by many manufacturers to denote standard pressure and ‘room temperature’, which is variously defined but is usually close to 23°C. Less ideally, a volumetric

Multiple-animal respirometry

The ideal multiple-animal metabolic measurement system provides continuous, accurate measurements of many animals simultaneously without any interruptions, including those for baselining. Thanks to recent advances in flow-through respirometry (e.g. Melanson et al., 2010; see Room Calorimetry section), it is now possible to do just that, but to date no laboratory in the world has implemented such a system for multiple animals, because it requires, ideally, two gas analyser chains per animal,

Room calorimetry

The accurate and long-term measurement of human metabolic rate is becoming an increasingly important part of understanding the epidemic of obesity now raging in most the world's industrialized nations (Vioque et al., 2010). Although mask flow-through systems – so-called “metabolic carts” – give adequate readings of humans tethered to the system by a mask, they are not suited to continuous use. To acquire a representative data set of untethered human metabolic rate, the standard instrument is

Direct calorimetry vs. flow-through respirometry

If there is a gold standard for measurements of metabolic rate in the laboratory, then the title is vied for by respirometry (indirect calorimetry) and direct calorimetry (the direct measurement of metabolic heat production). The latter is often considered a more accurate methodology because it bypasses certain assumptions/estimations made by respirometry when converting measurements of V˙O2 into power, such as the nature of the metabolic substrate and exclusive use of aerobic pathways (see

Closing notes and recommendations

As products of natural selection ourselves, and concerned with living systems that exist due to the process of evolution, we are learning to re-appreciate the fact that ‘since it is the intact and functioning organism on which natural selection operates… organisms are therefore a central element of concern to the biologist who aspires to a broad and integrated understanding of biology’ (Bartholomew, 1985). Metabolic rate is a fundamental, externally measurable characteristic of the whole

Acknowledgements

We thank Roger Seymour for his insightful comments on comparisons of direct and indirect calorimetry, as well as Pawel Koteja and two anonymous referees whose comments significantly improved our MS.

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    This submission is associated with a symposium held at the Society of Experimental Biology Annual Main Meeting 2010, Prague, entitled ‘The challenge of measuring energy expenditure: current field and laboratory methods’. Guest Editor is Dr LG Halsey.

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