LabAnalyst X    RESPIROMETRY
LabAnalyst X has seven menus, plus instructions and links in the Help menu and three functions in the LabAnalyst X menu.



This is a subset of the EDIT menu that covers calculations for respirometry and gas exchange.

Respirometry (the calculation of metabolic rate from changes in the concentration of two of the gases -- oxygen and carbon dioxide -- involved in cellular respiration) is simple in principle, but can be a real headache to do correctly.  The major pitfalls stem from the following issues:

  • Organisms both consume oxygen and emit CO2 -- but usually not in equal amounts.  This creates volume changes that must be accounted for. 
  • Air-breathing organisms also emit water vapor, which affects calculations by increasing total gas volume and diluting the concentrations of O2 and CO2. 
  • Some organisms emit (either anteriorly or posteriorly) substantial quantities of other gases -- such as methane -- that affect concentrations of O2 and CO2. 
  • Volumes of respiratory gases are strongly affected by temperature and pressure. 
  • The arrangement of the various elements of a respirometry system -- volume or flow meters, animal chambers, gas scrubbing units, and gas analyzers -- determines the form of the conversion equation that should be used. 
  • Rapid changes in an organism's gas exchange are buffered or 'hidden' by the mixing characteristics of the respirometry system.  To some extent, they also may be buffered within the organism:   for example, apparent CO2 emission can fluctuate with temperature changes, independent of metabolic events.
  • Some gas exchange calculations require knowledge of the concentrations of two gas species (usually O2 and CO2).  Unfortunately, these computations can be compromised because different kinds of gas analyzers respond at different rates.   This is of particular concern if metabolism is changing rapidly.
  • The energy equivalents of gas exchange rates differ between O2 and CO2, and also with the metabolic fuel being consumed. 

These issues -- and how LabAnalyst X deals with them -- are addressed in more detail below.

 Note: if you are a respirometry geek -- and who else would waste time reading this? -- you may have noticed that I'm not using the correct abbreviations for rates of oxygen consumption and carbon dioxide production. 'Proper' usage includes a dot over the V in VO2 and VCO2 to indicate rates instead of volumes.

     Alas, I haven't found a convenient way to make that 'Vee-dot' symbol in html.

  •    Open versus constant volume systems:   There are two methods in routine use for measuring rates of gas exchange: open system respirometry and constant volume ('closed system') respirometry.  In the former, a continuous flow of air (or other fluid) flows past the animal, and you measure the difference in gas concentration in incurrent vs. excurrent fluid.  Most of the calculations and discussion on this page concern open systems.

    In constant volume respirometry, the organism is placed in a sealed chamber, and over time its respiration changes the gas concentrations in the chamber.  You measure rates of gas exchange by determining gas concentrations (O2 and/or CO2) at the start and end of a period of measurement, and then using the cumulative difference in concentrations and the elapsed time to compute the average rate of change.  The most straightforward way to handle constant volume calculations with LabHelper and LabAnalyst X is as follows:

    A more versatile method for computing C.V.R. is in the SPECIAL menu. This offers more options than the C.V.R. window in the maximum value calculator

  •    With either open system or constant volume respirometry, if your animal is an air-breather you need to think about the possibility that its metabolism involves gases other than water, O2, and CO2.   For example, ruminants and other herbivores are notorious for producing large quantities of methane and other organic gases.  These result largely from the microbial fermentation of plant material in specialized regions of the herbivore's gut.  However, the effect is not limited to plant-eating species with specialized fermentation organs.  Predators such as snakes are known to emit substantial amounts of various decomposition gases during the digestive breakdown of prey.  These additional gases have two possible effects, both of which can compromise measurement accuracy:

    • With any gas analyzer, the presence of these gases will dilute the concentrations of O2 and CO2. Unless you can quantify this effect, you cannot calculate VO2 or CO2 accurately.  Fortunately, most animals don't emit enough of these gases to cause much of a dilution problem, but you do need to be aware of the potential.
    • With some oxygen analyzers that have high-temperature measurement cells (like the zirconia cells used by Applied Electrochemistry/Ametek S-3As), another error can be produced when organic gases oxidize -- combust -- in the cell.  The S-3A's cells operate at more than 500 °C, so there is plenty of potential for this to occur.  Oxidization removes oxygen from the gas stream.  It also produces CO2, water, and other reaction products that lower the concentration of the remaining O2 and further increase the error (generating an artifactually high VO2). 

    If you think your animal does produce substantial quantities of non-standard respiratory gases, the solution to both of these pitfalls is to remove the problematic gas species from analysis air by using an appropriate scrubbing filter upstream of the gas analyzer(s).   This, too, incurs operational penalties, such as reduced response time and more scrubbing tubes to keep fresh.

  •   COMPUTE VO2...   Y     
  •    COMPUTE VCO2...    G     
  •   COMPUTE EWL...       

    This sequence of windows converts gas concentrations (in % or optionally for EWL, in dew point temperature) into rates of gas exchange.  Results are stored either in the source channel or optionally (if the number of channels is less than 24) in a new channel labeled in appropriate units.  Flowrate is either entered directly in ml/min (from the source file or the keyboard) or obtained from another channel (i.e., readout from a flow meter).

    Gas calculations begin with flow rate source selection (in the PREFERENCES option in the FILE menu, you can select automatic lag and baseline correction prior to gas exchange calculations).

    Important:    The program expects all flowrates to be in units of ml/min.

    For VO2 and VCO2, you need to specify whether incurrent gas is dry and whether incurrent CO2 is absorbed, together with the concentrations of O2 and CO2 in incurrent gas (FiO2 and FiCO2; default values 20.95% and 0.035%, respectively).

    For EWL calculations, and for VO2 and VCO2 if you did not use dry incurrent gas, you will also need to indicate the operating temperature of the humidity sensor (this is done later; see below).

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    Channel selection:  Next you select the channel containing gas concentration data, and if you are obtaining flow from a channel, the flow rate channel (as shown below at left):

    Flow configurations:  Next you select a flow configuration ('Mode' 1, 2, etc.).  This specifies the conversion equation to be used, based on where flow rate is measured (upstream or downstream of the animal chamber and gas sensor) and how (and if) the the gas stream is dehumidified and scrubbed of CO2.  Options are shown graphically, as in the example at right.

    The 'masks' button (VO2 and VCO2 only) opens a similar window with a selection of mask configurations (example for VO2 shown at right).  A 'mask system' is defined as one in which unscrubbed ambient gas - usually air - is pulled past the animal to capture exhaled gas (i.e., gas is sucked past the animal under negative pressure).  In mask systems all pumps, flow meters, gas analyzers, etc.  are downstream.
           Some mask systems are functionally (and computationally) identical to some 'regular' flow configurations, and hence use the same conversion equations. The '?' button describes these overlaps, and also certain configurations to avoid if possible.

    Pick the configuration and mode closest to your own respirometry system.  It's quite important to give LabAnalyst X the right information about your flow arrangement.  In some -- but by no means all -- cases, with 'normal' incurrent concentrations of CO2 and O2 (about 0.035% and 20.9%), different modes may yield fairly similar results.  However, in some cases, serious errors (20% or more) can result if the wrong equations are used (this is most problematic for VO2).  Selecting the appropriate configuration is most critical if the sample gas from the animal chamber has large deflections from ambient concentrations of CO2 and O2.  An important and easily avoidable error will result if the gas stream at the flow meter contains a significant (but unknown) fraction of water vapor.  Therefore it's good practice to use DRY gas whenever possible -- and if you can't, be sure to compensate as described in the next section.

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    Compensating for water vapor:  In some flow arrangements, flow rate is measured on a 'wet' gas that is -- usually -- subsequently dried prior to measuring O2 or CO2 concentration (this includes modes 1 and 2 for VO2 and mode 1 for VCO2 [if incurrent gas isn't dry], and for most mask arrangements).  In such cases it may be necessary to compensate for the water vapor content.  It is also necessary to compensate for water content if O2 or CO2 concentration is measured with a 'wet' gas containing water vapor (see the compensating for sensor humidity section).  Compensation is necessary because water vapor affects gas volume.

    As can be seen in this diagram, the importance of water vapor compensation is greatest at high ambient temperatures and high humidities.  For example, at a temperature of 30 °C, saturated air (100% R.H.) is about 4 percent water vapor by volume.  That proportion increases to about 7 percent at 40 °C.

    You should also keep in mind that these values are for a total barometric pressure of 1 atmosphere.  Since saturation vapor pressure at a given temperature is constant regardless of the total pressure in the system, the fractional water vapor content increases as total pressure decreases.  For example, if you are working at an altitude of 2000 meters (where atmospheric pressure is roughly 590 torr), saturated air at 30 °C contains about 5.4% water vapor by volume.

    You can reduce or avoid these errors by using estimates of humidity, or preferably measurements of humidity or dew point, to calculate the volumetric fraction of incurrent gas comprised of water.  This figure should give you some idea of the need to compensate, given the temperature and pressure conditions you work with.

    Note that the temperature and pressures of importance are those at the position of the flowmeter, and not elsewhere in your system.  Clearly, if you are working at fairly low humidities and the temperature of your flowmeter is about 20 °C or lower, the expected error is on the order of 1% or less, and probably can be safely ignored.  Even in fairly warm conditions (around 30 °C), you can reduce the expected error to about 2% if you can guess relative humidity within 50%.

    Entering the required data involves several successive windows, the first of which is shown at right.  Note that it's necessary to adjust to ambient barometric pressure (even if flow rate has already been set to STP).  This is because the partial pressure of water vapor is constant at a given combination of relative humidity and temperature, regardless of the partial pressure of the rest of the gas mixture.  You also have the option of not compensating, if you wish.

    You are next asked for a channel containing humidity (or water vapor) data; alternately you can use a constant RH (the default is 20%).  This is shown below, at left.  Next you are next asked whether the humidity data are in units of relative humidity or dew point temperature (below, at right):

    •    If the data are in units of relative humidity, you are asked to select a channel containing humidity temperature data (as shown at right), or a constant temperature for the RH sensor: 

    The software will compensate for temperature differences between the humidity sensor and the flowmeter.

     One other effect of water vapor is (marginally) relevant if you use a mass flow controller or mass flowmeter to handle your flow rates.  These devices ("MF's") determine rates of gas flow by measuring rates of heat transfer.  Unsurprisingly, the thermal properties of water vapor are slightly different from those of dry air, so the addition of water vapor to the air stream will have an effect on the calibration accuracy of MF devices.  However, under most imaginable respirometry setups, this error is small and can safely be ignored.

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    Compensating for RQ:  One of the many troublesome complications in respirometry is that organisms seldom exchange just one gas at a time: instead, they are simultaneously consuming oxygen and producing CO2 (we'll leave water out of this discussion because -- usually -- it can easily be scrubbed from air streams prior to gas analysis).  Since the ratio of CO2 produced / oxygen consumed (the Respiratory Quotient, or RQ) is seldom exactly equal to 1.0, respiration causes a net change in the total gas volume.  As a result, gas concentrations are affected.  For example, consider a calculation of oxygen consumption:   If more oxygen is consumed than CO2 is produced, the total gas volume is slightly reduced, which results in an increase in the concentration of O2.  This seemingly trivial imbalance will influence exchange calculations and needs to be accounted for (startlingly large errors can result if you don't do this correctly).  The necessary equations can be derived from simple algebra, but are tedious in practice.  Fortunately, equations for most respirometry systems are built into LabAnalyst X (see below for details).

    If you are calculating oxygen consumption and you use a flow configuration that requires compensation for CO2 production, you are asked if you want to use a constant RQ value, or a previously computed channel containing VCO2 (note that VCO2 must be in units of ml/min, or you may get extremely inaccurate results), or CO2 concentration data (in % deflection from ambient CO2 concentration).  If you elect to use VCO2 or %CO2 , you will be asked to select the channel containing CO2 data.  For maximal precision, be sure to use the lag correction function to synchronize the timing of oxygen and CO2 data (especially important if there are large short-term fluctuations in gas concentrations).

    Similarly, if you are calculating CO2 production, you will always need to compensate for oxygen consumption (since animals rarely produce CO2 unless they are also consuming O2) and you are asked if you want to use a constant RQ value, or oxygen concentration data (in % deflection from ambient O2 concentration), or a previously computed channel containing VO2 in units of ml/min.

    In most cases, use of a constant RQ will produce quite good accuracy.

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    Units:  As a last step, you need to select the units for gas exchange.  In the final window of the sequence, you are presented with 4 or 5 edit fields containing mass, B.P., Ta, etc.  Adjust these as necessary.  You can select from a variety of conversion units in the pop-up menu (shown at right with ml/min selected).

    If you are calculating VO2 or VCO2 in watts or joules/time, you also need to set the correct value for heat equivalency (an example for a VO2 calculation is shown at right).  The defaults (20.1 J/ml for VO2 and 25.0 J/ml for VCO2 ) are reasonable for mixed diets.  Theoretical heat values are shown for pure carbohydrate, fat, and protein diets; see Gessaman and Nagy (1988, Physiol.  Zool.  61:507-513) for a discussion of gas exchange conversion factors for metabolic calculations.

    You can click buttons to select appropriate values for carbohydrate, lipid, or protein substrates.  It is possible to directly enter your own value into the edit field, however.

    You can show the conversion equation by clicking the 'show equation' button:

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    Conversion equations and options

    Equations used for computing exchange rates are derived in part from Depocas and Hart (1957; J.  Appl.  Physiol.  10:388-392), Hill (1972, J.  Appl.  Physiol.  33:261-263) and Withers (1977, J.  Appl.  Physiol.  42:120-123); others I derived myself.  The following symbols are used: FR = flow rate, V = exchange rate for the gas in question (oxygen, CO2, or water vapor), STP = factor for converting to standard conditions of temperature and pressure, Fi = input fractional concentration, Fe = excurrent fractional concentration, RQ = respiratory quotient.

     Note: if you have already done the STP correction to the a fixed flow rate or flow rate data in a separate channel -- for example, if you measured flow with a mass flow controller with STP-corrected output, or if you used the STP converter in the OUTPUT menu and saved the results-- make sure the temperature and pressure are set to 0 °C and 760 torr, respectively.

    ONE MORE CAUTION:  the algorithms used here are appropriate for most -- BUT NOT ALL -- respirometry systems.  Check to make sure your system exactly matches the conditions outlined below.

    For VO2, the calculations depend on the position of the flowmeter and whether (and where) water vapor and CO2 are absorbed.  Flow rate (FR) through the metabolism chamber is best measured upstream from the chamber in most cases.  If you need to humidify the air stream, the best arrangement is to use an upstream flow meter, with the air dried before it enters the flow meter and then re-humidified between the flow meter and the chamber.  For accurate calculations it is also necessary to remove water vapor and helpful to scrub CO2 from the air stream downstream from the animal chamber, before O2 content is measured.

     If both CO2 and water are scrubbed and the flowmeter is upstream from the chamber (Mode 1):

    VO2 = STP * (FiO2 - FeO2) * FR / (1 - FeO2)

    If both CO2 and water are scrubbed and the flowmeter is downstream (Mode 3; Mask mode 1 uses the same equation after correction of flowrate for incurrent water vapor content):

    VO2 = STP * (FiO2 - FeO2) * FR / (1 - FiO2)

    These are the simplest and most accurate ways to compute VO2

    For some flow configurations you need to know RQ for accurate calculation of VO2 , or you must have a previously computed channel containing VCO2 in ml/min, or have data on %CO2 in excurrent air (expressed as the difference from incurrent CO2 concentration).  If the animal is metabolizing carbohydrate, RQ is 1.0; if it metabolizes fat the RQ is about 0.7; if it metabolizes protein the RQ is about 0.8.  Mixed diets yield intermediate RQ.  If you don't know RQ or diet, the potential error is minimized if you use the default RQ of 0.85 (see Gessaman and Nagy 1988).  Also, you should be aware that there are situations where the CO2/O2 ratio can exceed 1.0 (for example, if the animal is depositing fat).

    CAUTION:   If you are using the 'calculate from %CO2' or 'calculate from VCO2' modes, you need to exactly synchronize the oxygen and CO2 channels in time.   This is especially important in serial configuration (first read CO2, then read O2) because the two analyzers are not 'looking' at the gas stream simultaneously (the lag correction option in the EDIT menu can usually fix this problem).   Note that if your subject's metabolism is changing rapidly, this may not be possible even if you split your sample gas stream and read O2 and CO2 in parallel, as the response times of different gas analyzers are usually unequal.   For example, the response time of a typical CO2 analyzer is seconds, while some O2 analyzers (like those from Applied Electrochemistry) respond in milliseconds.

     In 'calculate from constant RQ' mode:

    If the flowmeter is upstream and CO2 is not removed from the excurrent air stream at any point (Mode 2):

    VO2 = STP * (FiO2 - FeO2) * FR / (1 - FeO2 * (1 - RQ))

    If the flowmeter is downstream and CO2 is not scrubbed from the excurrent air stream prior to flow measurement but IS removed prior to oxygen measurement (Mode 4 or Mask Modes 2 and 4):

    VO2 = STP * (FiO2 - FeO2) * FR / (1 - FiO2 + RQ * (FiO2 - FeO2))

    If the flowmeter is downstream and CO2 is not removed from the excurrent air stream at any point (Mode 5 or Mask Modes 3 or 5):

    VO2 = STP * (FiO2 - FeO2) * FR / (1 - FiO2 * (1 - RQ))

     In 'calculate from VCO2' mode:

    If the flowmeter is upstream and CO2 is not removed from the excurrent air stream at any point (Mode 2):

    VO2 = STP * ((FiO2 - FeO2) * FR - FeO2* VCO2) / (1 - FeO2)

    If the flowmeter is downstream and CO2 is not scrubbed from the excurrent air stream prior to flow measurement but IS removed prior to oxygen measurement (Mode 4 or Mask Modes 2 and 4):

    VO2 = STP * ((FiO2 - FeO2) * FR + VCO2 * (FeO2 - FiO2)) / (1 - FiO2)

    If the flowmeter is downstream and CO2 is not removed from the excurrent air stream at any point (Mode 5 or Mask Modes 3 or 5):

    VO2 = STP * ((FiO2 - FeO2) * FR - FiO2 * VCO2)/ (1 - FiO2)

     In 'calculate from % CO2' mode:

    If the flowmeter is upstream and CO2 is not removed from the excurrent air stream at any point (Mode 2):

    VO2 = STP * FR * ((FiO2 - FeO2) - FeO2* (FeCO2 - FiCO2)) / (1 - FeO2)

    If the flowmeter is downstream and CO2 is not scrubbed from the excurrent air stream prior to flow measurement but IS removed prior to oxygen measurement (Mode 4 or Mask Modes 2 and 4); note that this mode is sensitive to whether CO2 is scrubbed from the incurrent air stream:

    VO2 = STP * FR * ((FiO2 - FeO2) - FiO2 * (FeCO2 - FiCO2) + FeO2 * FeCO2) / (1 - FiO2)

    If the flowmeter is downstream and CO2 is not removed from the excurrent air stream at any point (Mode 5 or Mask Modes 3 or 5):

    VO2 = STP * FR * ((FiO2 - FeO2) + FiO2 * (FiCO2 - FeCO2))/ (1 - FiO2)

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    For VCO2, you need to know RQ, or you must have data on oxygen content or consumption.  If you don't know RQ or diet, use the default RQ of 0.85, which usually produces results with an error of <5%.  Alternately, you can use a previously-computed channel containing VO2 in ml/min, or have data on %O2 in excurrent air (in difference from incurrent O2 concentration).  The equations (which assume gas is dry when FeCO2 is measured) are as follows:

    CAUTION:   If you are using the 'calculate from %O2' or 'calculate from VO2' modes, you need to exactly synchronize the oxygen and CO2 channels in time.   This is especially important in serial configuration (first read CO2, then read O2) because the two analyzers are not 'looking' at the gas stream simultaneously (the lag correction option in the EDIT menu can usually fix this problem).   Note that if your subject's metabolism is changing rapidly, this may not be possible even if you split your sample gas stream and read O2 and CO2 in parallel, as the response times of different gas analyzers are usually unequal.   For example, the response time of a typical CO2 analyzer is seconds, while some O2 analyzers (like those from Applied Electrochemistry) respond in milliseconds.

     In 'calculate from constant RQ' mode:

    If the flowmeter is upstream from the chamber (Mode 1 or Mask Mode 1):

    VCO2 = STP * (FeCO2 - FiCO2) * FR / (1 - FeCO2 * (1-(1/RQ)))

    If the flowmeter is downstream from the chamber and CO2 is not scrubbed prior to flow measurement (Mode 2):

    VCO2 = STP * (FeCO2 - FiCO2) * FR / (1 - FiCO2 * (1-(1/RQ)))

    If the flowmeter is downstream and CO2 is scrubbed prior to flow measurement (Mode 3 or Mask Mode 2):

    VCO2 = STP * (FeCO2 - FiCO2) * FR / (1 - FeCO2 + FiCO2/RQ)

    If you are using a mask and the flowmeter is immediately downstream from the animal, and water is scrubbed prior to CO2 measurement (Mask Mode 3):

    VCO2 = STP * (FeCO2 - FiCO2) * FR / (1 - FiCO2 * (1 + 1/RQ))

     In 'calculate from VO2' mode:

    If the flowmeter is upstream from the chamber (Mode 1):

    VCO2 = STP * (FR * (FeCO2 - FiCO2) - FeCO2* VO2) / (1 - FeCO2)

    If the flowmeter is downstream from the chamber and CO2 is not scrubbed prior to flow measurement (Mode 2 or Mask Mode 1 or Mask Mode 3):

    VCO2 = STP * (FR * (FeCO2 - FiCO2) - FiCO2* VO2) / (1 - FiCO2)

    If the flowmeter is downstream and CO2 is scrubbed prior to flow measurement (Mode 3 or Mask Mode 2):

    VCO2 = STP * (FR * (FeCO2 - FiCO2) - FeCO2* VO2) / (1 + FeCO2)

     In 'calculate from % O2' mode:

    If the flowmeter is upstream from the chamber (Mode 1):

    VCO2 = STP * FR * ((FeCO2 - FiCO2) - FeCO2* (FiO2 - FeO2)) / (1 - FeCO2)

    If the flowmeter is downstream from the chamber and CO2 is not scrubbed prior to flow measurement (Mode 2 or Mask Mode 1 or Mask Mode 3):

    VCO2 = STP * FR * ((FeCO2 - FiCO2) + FiCO2* (FiO2 - FeO2)) / (1 + FiCO2)

    If the flowmeter is downstream and CO2 is scrubbed prior to flow measurement (Mode 3 or Mask Mode 2):

    VCO2 = STP * FR * ((FeCO2 - FiCO2) + FiCO2* (FiO2 - FeO2)) / (1 + FeCO2 - FiCO2)

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    For Evaporative Water Loss, the program assumes there is no gas volume change between the part of the system where flow rate is measured (whether upstream or downstream of the chamber) and the point where water content is measured.  The equation is:

    EWL = (FeH2O - FiH2O) *STP * FR

    with appropriate correction for whatever output units are desired.  This ignores any effects of VO2, VCO2, and changed water vapor content on flow rate (in combination, these exchanges usually have a very minor effect on calculated EWL).  The equation is simple, but figuring out FiH2O and FeH2O from typical humidity data is not, because of the non-linear relationship between temperature and the water content of a gas. 

    Water vapor content is usually measured as either percent relative humidity or dew point temperature (in °C).  Select the appropriate units for your humidity sensor's output.  In either case, values must be converted into water vapor densities and then into fractional concentrations.  The equations for computing water vapor density are arithmetically rather nasty (and therefore the speed of conversion isn't as fast for some EWL calculations as for other kinds of gas exchange).  If you use % RH, the algorithms need to know the temperature of the humidity sensor.  You can either enter this directly or the value can be obtained from a data channel.  In the latter case, and for all analyses of dew point data, vapor density calculations must be repeated for each sample point.  That slows the rate of conversion considerably, so a 'bar graph' display is shown to indicate progress.

    The algorithms used to compute vapor density are derived from Properties of Air, by Tracy, Welch, and Porter (1980; University of Wisconsin).  Accuracy should be ±0.4% or better at temperatures between -20 and 70 °C.  Note that in practice it is very difficult or impossible to avoid error-inducing condensation or freezing of water vapor when working at subzero temperatures.

    Miscellaneous notes for gas exchange:

    The 'instantaneous' correction helps compensate for the volumetric washout characteristics of respirometry systems, using the 'Z' correction approach modified for exponential washout kinetics (Bartholomew, Vleck, and Vleck 1981, Journal of Experimental Biology 90, 17-32).  It works best in systems with large flow rates relative to effective volume.  In general, it is prudent to avoid this somewhat rude manipulation of data if the system volume is large and the flow rate is low, unless the chamber is very well stirred (as in a recirculating wind tunnel or a chamber with mixing fans).  In particular, be aware of the following:

    NOTE:  Movements of animals within chambers, relative to the position of input and output ports, can introduce very ugly errors in instantaneous calculations because of transient artifactual enrichments or rarifactions of local gas concentrations that have nothing to do with real metabolic rates.

    Basically, the instantaneous conversion algorithm works from estimations of how rapidly a gas concentration that has been transiently deflected from a constant value will return to a state of zero deflection if no other changes occur.  Another way of putting it is as follows: if an animal instantly changes its metabolism to a new steady-state value, how long does it take for the excurrent gas concentrations to achieve their new steady state values? An instantaneous change in metabolism will be detected at the gas analyzer as a gradual approach to new steady-states, as shown schematically here:

    What the 'instantaneous' conversion does is back-calculate from the measured concentration changes to approximate the real event:

    For VO2 calculations the algorithm assumes incurrent oxygen concentration is 20.95% and offset so as to read zero at 20.95% oxygen.  For carbon dioxide and water vapor it assumes that incurrent concentrations are zero % (not offset).  Calculations will yield inaccurate results if these assumptions are violated.  The so-called effective volume (below) is necessary for these calculations.  It can be derived from a washout curve for the system being used.

    The response correction option in the TRANSFORMATIONS routines (EDIT menu) can also adjust for mixing problems and washout characteristics.  It is based on an unmodified Z-transform, and in some cases may be easier to use than the instantaneous correction.

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  •   SENSOR HUMIDITY COMPENSATION...    Normally, the preferred procedure is to scrub water vapor from the sampled gas stream before measuring the concentrations of O2 or CO2.  However, in some respirometry systems it is necessary or desirable to analyze 'wet' gas -- i.e., gas that contains some water vapor.  For example, you might want to make uninterrupted measurements over a very long period.  This introduces a potential problem: any desiccants used to scrub water vapor may be exhausted before the measurements are completed (and you won't be able to tell which data were obtained from dry gas and which were not).  By not removing water vapor at all, you can avoid that complication.

    In order to accurately compute gas exchange rates from undried gas, it is necessary to remove the dilution effect of water vapor in the gas stream (which slightly reduces the concentration of O2 or CO2 in the gas analyzer).  This causes an underestimate (VCO2) or overestimate (VO2) of 'true' gas exchange rates.  The resulting error is small if the temperature and relative humidity of the measured gas are low, but increase rapidly as temperature and humidity climb (see the figure in the section on water vapor correction showing the volumetric contribution of water vapor at different temperatures and humidities).  Low barometric pressure increases the error still further.

    The Sensor humidity compensation option lets you use several methods for compensating for measurements on wet gas, but for all of them you must provide information of the temperature and water content of the measured gas stream.  You can use recorded channels of relative humidity or dew point, or provide a constant value.  Similarly, you can use recorded or constant values for temperature.  These are selected through a series of windows.  Note that some combinations require extensive vapor pressure calculations for each data point, which slows the computation speed; in these cases a progress bar is shown.

    Note that these routines compensate only for the passive dilution effect of water vapor.  They cannot rectify any active interference with the sensor caused by water.  In other words, if a sensor detects water vapor and 'confuses' it for the gas of interest (CO2 or O2), there is no alternative to completely drying the gas stream prior to measurement. Fortunately, most O2 and CO2 analyzers are relatively immune to this problem.
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  •   VO2 IN WATER...   This routine will calculate oxygen depletion curves or rates of O2 consumption of acquatic organisms, based on changes O2 concentration. The latter can be expressed in a variety of units: oxygen partial pressure, i.e., pO2 (torr), percent saturation of oxygen (zero to 100%), parts per million O2 (by mass), or mg O2/Liter (note that the latter two units are identical).

    • For closed-system measurements, LabAnalyst estimates the amount of dissolved oxygen in the container at the start of measurements from container volume, temperature, solute concentration, initial oxygen saturation, and the partial pressure of O2 in the gas phase (calculated from barometric pressure and the percentage of O2 in the gas mixture, and accounting for the water vapor content of air at 100% RH). 
    • For open-system measurements, LabAnalyst estimates the amount of dissolved oxygen in the water flux through the system from temperature, solute concentration, initial oxygen saturation, and the partial pressure of O2 in the gas phase (calculated from barometric pressure and the percentage of O2 in the gas mixture, and accounting for the water vapor content of air at 100% RH).  Flow rates are obtained either from a channel or from keyboard entry (you can also specify whether deflections in oxygen content are positive or negative). 

    Most of these parameters can be edited with the EDIT FILE DATA option, including flow rate, barometric pressure, temperature, and container volume (= effective volume).  Alternately, you can enter your own O2 content data (dissolved oxygen per unit volume) and click the 'net O2' button, or you can directly enter net O2 content (total dissolved oxygen).

    If there are less than 24 channels in the file, you can store results in a new channel.

    For closed-system measurements, there are three conversion options.  For any of these, you can generate absolute units or mass-specific units (per mg, per g, or per kg).  Note that all conversions are based on the value shown in the 'net O2 content' edit field.  If you know that oxygen content differs from the computed value in this edit field, you can enter your own value prior to doing the conversion. 

    • cumulative oxygen content. This simply converts the input data (% saturation, pO2, ppm, mg/L) into the residual oxygen content of the container.  You can select several different output units, such as mL, microliters, mmoles, or micromoles.  The slope of this line over time (multiplied by -1) is the average rate of oxygen consumption (VO2). 
    • total oxygen used.  This option computes the cumulative amount of oxygen used during the measurement, again with a choice of output units.  The slope of this line over time is the average rate of oxygen consumption.  As for the previous option, several choices of units are available. 
    • oxygen consumption rate. This computes the rate of oxygen consumption (VO2; in a choice of units) by taking the point-to-point derivative of the relationship between oxygen use and time.  Note: unless the input data have been highly smoothed, results may be noisy when averaged over short time intervals.  However, long-term VO2 averages should be quite accurate, provided the O2 consumption of the organism was stable over time. 

    Here is an example showing conversion into consumption rate (ml O2 / [g .  min]):

    The Open-system (flow-through) routine will only compute oxygen consumption rate, in your choice of units.  You can use either of two algorithms, selected with buttons in the initial window:

    • In absolute value mode, input data are assumed to be absolute values, and VO2 is calculated by subtracting them from baseline O2 concentration (i.e., the amount of O2 in the incurrent water stream).  Baseline O2 concentration is computed from pressure, temperature, etc.  as described above.  For example, if baseline O2 concentration is 100% saturated, an oxygen saturation datum of 1% is assumed to mean that 99% of the oxygen in the water stream has been consumed, and VO2 is computed accordingly. 
    • In change from ambient mode, baseline O2 concentration must be offset to zero (this should be done with the BASELINE option prior to oxygen calculations).  In other words, an oxygen saturation datum of 1% is assumed to be a 1% change from baseline percent, not an absolute value of 1%. 

    Deflections in O2 concentration in the excurrent stream may be either positive or negative (if the latter, click the 'negative deflection' button in the initial window).

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  •    O2 HEAT EQUIVALENCE...

  •    CO2 HEAT EQUIVALENCE...   These options let you set the caloric equivalent of one ml of oxygen or CO2; the defaults are 20.1 joules/ml O2 and 25.0 joules/ml CO2. You can click buttons to select appropriate values for carbohydrate, lipid, or protein substrates.  It is possible to directly enter your own value into the edit field, however.

    This window appears automatically during gas exchange calculations if you select output units of energy instead of gas volume.

  •    EFFECTIVE VOLUME...   (Active channel only) Uses a recorded gas washout curve to compute the 'effective volume' of an open-circuit respirometry system.  Effective volume is an expression of the ratio of system volume to flow rate (it corresponds only approximately to the real volume of chamber and plumbing).  This routine bases its calculations on washout rates of any gas, as long as the washout deflection is measured as a change in % concentration and the equilibrium concentration is offset to a 'baseline' of zero.  This is done either during measurement or with the baseline correction and transformations routines in LabAnalyst.

    To generate a washout curve, set up the respirometry system as it is normally used (but without an animal) at the flow rate used for measurements.  Make a recording of gas concentration, starting at equilibrium levels.  After establishing a baseline, deflect concentration by rapidly exhaling into the incurrent flow upstream from the chamber (or quickly inject a bolus of some gas with different concentration than reference gas).  Continue recording as gas concentration rapidly peaks, slowly declines, and eventually returns to equilibrium concentration.  Save the data.  In LabAnalyst, correct the baseline to zero, even for oxygen files (it will probably help to smooth the data also).  Mark a block in the washout curve as it returns to equilibrium.  Usually it is best to select from the middle of the washout curve.  Then select the effective volume option.  LabAnalyst will request the flow rate and compute effective volume.

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    Flow rate measurement accuracy

    In open-system respirometry, the most frequent source of error is probably inaccuracies in measurement of gas flow rate.  Aside from the required correction to standard temperature and pressure (STP; 0 °C, 760 torr), there are several common problems that can compromise accuracy:

  •    An inaccurate calibration of the flow measuring instrument.  Most of the various devices used to determine gas flow rates -- turbine-based meters, rotometers ('ball in a tube' meters), mass flow meters, etc.  -- require periodic recalibration to maintain measurement accuracy.  Even if the initial factory calibration is dead on, there will be some drift over time.  Note that this is particularly true for mass flow devices (both meters and controllers).  After extensive use, the inevitable slight buildup of airborne contaminants on the sensor will cause calibration drift.  There may also be some gas leakage or pressure change in the reference portion of the sensor system.

    • Recalibration can be done in a number of ways.  You can send your units back to the factory (at considerable cost in time and expense).  Alternately, you can check your meter against another meter with a calibration you trust.  Better yet, you can calibrate against a volume meter (like a bubble meter or a good dry volume meter) that is relatively immune to calibration drift (be sure to correct for STP during the calibration process).
               One good do-it-yourself calibration method, particularly useful with dry-volume meters, is to evaporate a carefully weighed quantity of liquid nitrogen or dry ice (CO2) through the meter, and then checking the cumulative volume against the expected volume of the evaporated gas (22.4 liters per mole, at STP).   Be sure to account for effects of air pressure (if different from 760 Torr) and temperature (if different from the meter's calibration temperature).

  •   Working at unusual altitudes or pressures.  Most flow meters are designed and calibrated for a specific pressure range, usually fairly close to standard sea-level air pressure.  Usually, modest deviations from that standard have little effect on accuracy, but if you go to high altitude (several thousand meters) or work in strongly hyperbaric conditions, you should check your calibration.  Even some mass flow devices are not immune to this problem (although they are supposed to compensate for the effects of pressure changes).

    • The accuracy of devices that measure gas volume changes directly (bubble meters and dry volume meters) is generally unaffected by changes in working pressure (at least within the range of pressures tolerated by most air-breathing organisms). 

  • Use of unusual gas mixtures.  Most flow devices are calibrated for a specific gas (or gas mixture) and may require a correction factor when used with different gases.  Generally, for the kinds of devices used by people doing respiromentry, the factory calibration is for pure nitrogen or for air.  As an example of the effects of non-standard gas mixtures, consider 'helox' (21% oxygen, 79% helium).  This mix is often used by physiologists to induce cold stress without risk of freezing injury, or to alter the diffusion and viscosity properties of the breathing mixture (it also makes you talk in a squeaky voice if you breath it).  If you use helox with a mass flow device calibrated for nitrogen or air, the real flow rate is about 33% greater than the indicated flow (this is because mass flow devices measure flow from rates of heat transfer, and the heat transfer properties of helium are quite different from those of nitrogen).

    • Devices that measure gas volume changes directly (bubble meters and dry volume meters) are essentially unaffected by different gas mixtures. 

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