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