## John R. B. Lighton

Print publication date: 2008

Print ISBN-13: 9780195310610

Published to Oxford Scholarship Online: September 2008

DOI: 10.1093/acprof:oso/9780195310610.001.0001

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# Coulometric Respirometry

Chapter:
(p.18) 3 Coulometric Respirometry
Source:
Measuring Metabolic Rates
Publisher:
Oxford University Press
DOI:10.1093/acprof:oso/9780195310610.003.0003

# Abstract and Keywords

This chapter describes the theory and practical applications of coulometric respirometry. Coulometric respirometry is probably the most accurate method for measuring oxygen consumption rates. It is ideal for small animals and has the dual advantages of high sensitivity and the fact that the oxygen in the organism's environment is not depleted, allowing measurements to continue for long periods in many cases. The technique works by maintaining a constant pressure in a sealed system by electrolytically producing oxygen at the same rate at which an enclosed organism consumes it.

Coulometric respirometry is an elegant but seldom used technique. It is based on constant volume and constant pressure in a sealed respirometer chamber and is remarkable on two grounds: its excellent accuracy and its potential for making long-term measurements on suitable organisms.

As discussed in the previous chapter, the maximum duration of a respirometric measurement using a closed respirometer chamber, assuming CO2 absorption, is limited by the amount of O2 available and the rate at which the enclosed organism consumes O2. Coulometric respirometry sidesteps this issue by sensing the decline in pressure caused by consumption of O2 within the chamber and generating O2 in quantities sufficient to hold the pressure constant. For small animals, especially, electrolytic generation of O2 works well, and it has the advantage that a known charge produces a precisely known molar quantity of O2.

Coulometric respirometry was first described by N. T. Werthessen (1937), who used it to measure rat metabolism. Subsequent authors have used it chiefly for smaller organisms; excellent treatments can be found in Heusner et al. (1982) and Hoegh-Guldberg and Manahan (1995). Here I concentrate chiefly on small-animal use.

To generate O2, a saturated solution of copper sulfate (CuSO4) is electrolyzed using a platinum anode and a copper or platinum cathode. The electrochemical equation is

(3.1)
$Display mathematics$

Oxygen is generated at the anode, which should be sharpened to facilitate the production of tiny bubbles, while copper slowly plates onto the cathode. The volume of O2 released is

(3.2)
$Display mathematics$
where Q is the charge of electricity discharged into the solution in coulombs, V m is the molar volume of O2 at STP (22.413 × 109 nl), and F is the Faraday constant (96,485 coulombs · mole−1).1 You must therefore try to apply a precisely known charge into the electrolyte to generate a known quantity of O2. Fortunately this is quite easy to do. The charge stored by a capacitor is precisely CV, where C is capacitance in farads (p.19) and V is the voltage stored by the capacitor, assuming that it is discharged to 0 V. This is not advisable in the case of electrolysis, because CuSO4 will not decompose electrolytically below an applied potential of 2 V. Thus, you must charge a capacitor to some initial voltage, V i, which is significantly greater than 2 V, stop the discharge at V s, which is slightly greater than 2 V, and multiply by the coulometric constant (58,073) to obtain the volume of O2 released per partial capacitor discharge:
(3.3)
$Display mathematics$

High-quality capacitors with precisely known values are easy to obtain (see appendix), and they remain highly stable over time. The steps are

1. 1. Measure the pressure differential between the respirometer chamber and an adjacent thermobarometer.

2. 2. When the pressure in the respirometer falls below some set point, discharge a capacitor from V i to V s through a pair of platinum electrodes in a chamber, adjacent to and connected to the respirometer chamber, containing CuSO4 electrolyte.

3. 3. Repeatedly discharge the capacitor, and count the number of discharges until the pressure in the respirometry chamber reaches the set point again.

It is then easy to compute the volume of O2 added to the chamber, using the above equations. Plainly, the system has two critical components, and these are the means of sensing pressure differentials and of discharging the capacitor accurately and repeatably into the electrolyte.

Unlike the differential pressure sensor described in chapter 2, the pressure sensor used for coulometric respirometry does not need to be either linear or accurate because it is simply a threshold sensor that operates in a closed-loop control system. It does, of course, need to be stable. Under these loose selective constraints, several different pressure sensors have evolved.

Perhaps the most complex (but also the most precise) is that described by Heusner et al. (1982) and later elaborated by Hoegh-Guldberg and Manahan (1995). Basically, a column of saturated CuSO4 in water in a glass tube senses the differential pressure between the respirometer chamber and the thermobarometer. Within the glass tube a Teflon ring constrains the meniscus on the respirometer chamber side, and pointing down into the meniscus is an electrolytically sharpened platinum electrode. Ingeniously, this electrode is also the anode of the electrolysis system. When the electrode makes contact with the CuSO4 solution, a circuit is completed, triggering a second circuit to discharge a capacitor through the electrode into the solution, releasing O2. A succession of discharges occurs until the accumulated O2 drives the meniscus back and breaks the contact (fig. 3.1). It's important to note that the roles of the platinum electrode as a contact sensor and as an anode are separate; the two operations do not occur simultaneously. The meniscus—Teflon interface is one of exquisite sensitivity, as demonstrated via trigonometric analysis by Heusner et al. (1982). Suitable circuitry for implementing the capacitor discharge technique is described by Heusner et al. (1982); Hoegh-Guldberg and Manahan (1995) describe (p.20)

Figure 3.1. A coulometric respirometer conceptually similar to those described by Heusner et al. (1982) and Hoegh-Guldberg and Manahan (1995). The respirometer is enclosed in a thermobarometer (TB), which is normally placed in a waterbath. The O2 consumption of either aquatic (AQ) or terrestrial (T) organisms can be measured. CO2 produced by the organisms is absorbed by a scrubber (C). As O2 is consumed, the electrolyte (E) rises up to the Teflon washer (TW) and touches the anode (V+), completing the electrical circuit and allowing electrolysis to take place, producing O2 in pulses created by repeatedly discharging a capacitor until the electrolyte meniscus is driven out of contact with the anode. The number of discrete pulses over time, each corresponding to the production of a known and fixed amount of O2, allows O2 consumption rate to be calculated.

the full implementation of such a system, including computer-assisted data acquisition and analysis.

A number of potential sources of error exist in coulometric respirometry, though their predicted magnitude is small (Heusner et al., 1982). Hoegh-Guldberg and Manahan (1995) looked with great thoroughness at the actual magnitude of the errors in practice, expressed as deviation in actual O2 production from theoretically expected values. To assess actual O2 production, they used both volumetric and actual analytical O2 measurements, and in each case they assessed production rate using least-squares regression analysis with the number of discharges on the X axis. The theoretical volume of O2 per discharge in their system was 4.20 pmol. Using volumetric techniques, they measured a value of 4.19 pmol per discharge, and using direct O2 analysis, they measured a value of 4.18 pmol per discharge. In the hands of careful workers, this technique is plainly capable of outstanding accuracy.

A simpler yet in some ways more versatile approach is described in detail by Tartes et al. (1999), in a design adapted from Sláma (1988). Here, the pressure sensing (p.21) and the electrolysis are separated. A thin capillary of ethanol senses pressure, and its position is determined photoelectrically by an LED/phototransistor gate (fig. 3.2).

Photoelectric detection of meniscus position is capable of great precision, yet it is mechanically simple to set up and noninvasive. Sláma (1988) used a sensitive differential pressure transducer instead of an ethanol meniscus, but the key principle remains equivalent; either technique is intrinsically analog rather than digital (on/off), and this offers an important advantage over the electrical contact technique. The excursion of the meniscus through the light gate (or the deflection of the diaphragm of the differential pressure transducer) gives rise to a range of electrical values that can be used to determine how far the pressure is away from a given set point. Thus, because the pressure can now be sensed over a range of values (as opposed to

Figure 3.2. An alternative, continuously recording coulometric respirometer design, extensively simplified from Tartes et al. (1999). A thin capillary manometer (M) interrupts a light beam between a photoemitter (E) and a photodetector (D) as the experimental organism, whether aquatic (AQ) or terrestrial (T), consumes O2. A CO2 scrubber (C) absorbs CO2 from the experimental organism and maintains a set relative humidity (see text). A graded current, proportional to manometer displacement, flows into a sharpened platinum anode (a copper cathode is used). The current passing through the electrolyte is proportional to the displacement of the ethanol meniscus relative to the light gate; the greater the displacement, the greater the current, and the greater the counteracting production of O2. The current passing through the electrolysis system is recorded and can be calibrated in O2 consumption rate, allowing continuous records to be kept. A thermobarometer (TB) reduces sensitivity to barometric pressure and temperature changes.

(p.22) in a binary, on/off fashion as with Heusner et al., 1982), an important new development is possible. The further the chamber pressure drops below its set point, the more current that can be passed through the electrolytic solution in a closed-loop control system, thus generating proportionately more oxygen to push the chamber pressure back to its set point. By recording the current delivered to the electrolysis system, this arrangement essentially gives a continuous readout of O2 consumption rate. All that is needed is the conversion factor to convert from current to STP-corrected O2 volume. This conversion factor is 209.5 μL O2·mA−1·h (Taylor, 1977). Using this system, Tartes et al. (1999) were able to measure O2 consumption rates down to about 0.2 μL·h−1 on a continuous basis, provided the anode was sharpened optimally so that the requisite tiny bubbles could be generated.

At present, there is no work of which I am aware involving coulometric respirometry of larger animals such as vertebrates. It can be argued that there are practical and logistical reasons for this, though it should be pointed out that large-scale coulometric production of O2 is hardly infeasible; for instance, it is routinely used in manned spacecraft. The real reason lies in the fact that metabolic data can be far more easily obtained in other ways, in the case of large animals at least, and I explore these approaches later, especially in chapters 10 and 11.

That said, coulometric respirometry is by far the most sensitive (at least semi-) mainstream technique for measuring O2 consumption in small animals, particularly over long intervals. It deserves to be more widely used. As Heusner et al. (1982) point out,

The present use of coulometric methods for metabolic studies does not do justice to their considerable advantages over the conventional gasometric methods. In particular, they are ideally suited for automatic long-term recording of instantaneous O2 consumption in very small animals. Their calibration is very stable in time and independent of the geometry of the respiratory chamber. The respirometer can be autoclaved, and the electrolytic O2 is sterile; therefore the recording of O2 consumption in tissue or organ cultures is possible under aseptic conditions and over extended periods of time. (p. 185)

Humans are conservative beasts and, being human, so are scientists. The primary factor preventing the wider use of coulometric respirometry is simply that commercial setups are not yet available. Perhaps this will change in the future and this promising technique will become more accessible and popular.

## Notes:

(1.) My maternal great-great-grandfather, Sir William Thomas Brande, who is now entirely forgotten, used to give public lectures at the Royal Institution in London during the 1850s that were set up by Michael Faraday who, according to a contemporary chronicler, “as-sisted Mr. Brande in his lectures, and so quietly, skillfully, and modestly was his work done, that Mr. Brande's vocation at the time was pronounced 'lecturing on velvet.’”