PaCoS Exercise 2b: ‘Spirometry’

Quantitative Effects of Inspired O2 and CO2 on the Minute Volume in the Resting Human

Overview

This practical runs over two laboratory sessions; the overall aim is to vary (and measure) O2 and CO2 in inspired air and see how these affect ventilation (as measured by the Minute Volume - the volume of air breathed out in one minute) in a population of non-exercising students - ie you!
Since we will be measuring the concentrations of both gases (the independent variables) as well as the subjects' ventilation (the dependent variable), we will be able to define in a quantitative way how ventilation is controlled by these gases (by using regression analysis).

You will use the first session to formulate and become familiar with the experimental methodology.

The second session will be used to do the 'real' experiment - collecting sets of data suitable for analysis. We will improve the power of that analysis by giving each of you an additional set of 300 readings to add to your own group's data – so, if you choose to write up this experiment*, you should not write your report based just on the data you collect in the lab, you should combine it with your own unique set of data that can be downloaded via

*You must write up either this experiment or the 'lactase' experiment as an individual report written in the style of a scientific paper/report, to be submitted early in the second semester.

Preliminary Experiments (session 1)

The main point of the first session is to get you familiar with the methodology (so you can minimise experimental errors) and have confidence that the gas concentrations you are using produce measurable changes in Minute Volume.

  • Can you set up and balance the spirometer?
  • Can you load the spirometer with a specific gas combination(eg High CO2 and Low O2)?
  • Can you correctly use the gas analyser?
  • Can you raise the subject's resting Minute Volume by raising CO2 and/or lowering O2? (see next week’s protocol, below, for ways to do this)
  • Can you accurately measure Minute Volume from the recording of spirometer movements?

Setting up/Calibrating the Spirometer

  • Launch LabChart and open the settings file “spirometry” from the Experiments tab in the Welcome Center.
  • With the pipe at the top of the side canister disconnected, gently move the spirometer wedge up and down to ensure it moves freely (check the pivot points) and the trace appears on the computer
  • Raw output from the spirometer’s transducer is measured in millivolts (mV). It needs to be calibrated to display the more meaningful units of litres (L). You can calibrate the trace by capturing a recording with the scale on the wedge at 0 litres and then at, say, 8 litres; you can then highlight this ‘step-trace’ and go through the standard ADI calibration routine as detailed below...
  • Start recording whist the wedge is set to empty (0 litres). Record for 10 seconds, then move the wedge up to 8 litres and record for a further 10 seconds, then Stop (do not Stop between the 0 and 8 portions of the recording).
  • Click-and-drag to select representative data both sides of the ‘step’. Select Units Conversion from the Channel 1 Channel Function pop-up menu (on the right side of the screen, next to the channel’s trace). This will display the following…

  • Select a representative section of the data when the wedge was set at 0 litres, and click the arrow next to “Point 1”. Type 0 in the adjacent box (see arrow)
  • Select a representative section when the wedge was set at 8 litres, and click the arrow next to “Point 2” (see above). Type 8 in the adjacent box.
  • Select litres from the units drop down menu.
  • Click Apply and then OK.
  • Now the calibration is done, move the counterweight to a position along its bar so that the wedge balances fairly well when about half full.

Tips

Spirometer

  • Balance the spirometer with the elephant tubing disconnected from the soda lime container.
  • The exhaust side of the mouthpiece/one-way valve set should be connected to the elephant tubing that goes first to the soda lime container.
  • When you use soda lime, use all of it!
  • Soda lime changes colour when it no longer works.
  • Don't fill the spirometer from the natural gas tap!!!
  • Rinse mouthpieces off before use (to remove the disinfectant); do not return used mouthpieces to the container containing the rest of the fresh ones!

Subject

  • Tilt the mouthpiece slightly up when it's in the mouth (to stop dribble running into the valve set).
  • Don't forget to put on a nose-clip.
  • Do not look at the recording during the experiment (it may influence the recording).
  • Do not involve yourself with the experiment - just sit there quietly!
  • Try not to think of your breathing (difficult with a mouthpiece in!)
  • If you feel uncomfortable at any time - discontinue the recording/experiment. This is not a test of machismo/endurance.

Gas Analysis

  • When sampling air in the spirometer with a gas syringe, discard the first 100 ml sample, then immediately re-sample and analyse that second sample (this eliminates errors due to whatever air is in the sampling pipe).
  • Carry the syringe around flat with a finger over the nozzle.
  • Don't push air into the gas analyser; it has a built-in pump and sucks the samples in itself.
  • Take a pen and paper to the gas analyser - don't rely on memory!
  • Don't empty the spirometer until both gas concentrations have been measured and recorded - just in case you need to sample again!

Recording

  • Ensure the ADI software has been calibrated so that measurements of the vertical deflections on the recordings are expressed in litres.
  • 'Mark' the recording at the point at which the gas sample to be analysed is taken.
  • Ensuring that the experiment and the recording continues for at least 30 sec after taking the gas sample; you can then pause the ADI software (when you do the real experiment you will end up with nine 'blocks' of recording corresponding to each gas combination) and allow the subject to disconnect.
  • Make a note of the sequence of gas combinations you use - it is obviously vitally important that each recording is assigned to the correct gas combination!

The Experiment (to be done in session 2)

You should aim to take at least one subject from your group through the nine different gas combinations (it is better to do one subject well than two badly).

Bear in mind the following points...

  • You don't have to work through the gas permutations in a particular sequence, in fact a random sequence is best (however, you may wish to do all of the experiments requiring soda lime back-to-back)
  • Make a note of the height (cm) and weight (kg) of each subject (for the later calculation of body surface area)
  • Remember to discard the initial sample when taking a gas sample for analysis (this is the single most important point when it comes to getting good data!)
  • Measure the Minute Volume across the one-minute segment of the spirometer recording with the marker showing when the gas was sampled in the middle (you should have 'marked' the recording at, or noted, the precise time at which the sample was taken)
  • Ensure your recorded data make sense (and are legible). The MV at rest is about 6 litres so, if your measurements suggest less than this you should check your calculations (or the equipment for leaks)! Expect MV to increase to > 50 litres at high carbon dioxide/low oxygen
  • Make enough copies of the data for all in your group. Your will need to add these data to your own unique dataset that will be provided for you (see should you choose to write up this exercise.

Suggested protocol…

Low CO2 / Med CO2 / High CO2
Low O2 / Air
Soda lime
3 min / Air
2 min / Air
3 min
Med O2 / Air*95/5
Soda lime
3 min / Air* & 95/5
2 min / Air* & 95/5
3 min
High O2 / 95/5*
Soda lime
3 min / 95/5*
2 min / 95/5*
3 min

* There are three ‘compartments’ in the experimental system; the gas in the spirometer wedge, the gas in the elephant tubing, and the gas in the subject. So, filling the spirometer wedge with 95/5 and then doing the experiment will actually result in dilution of the oxygen to a ‘medium’ level (around 40%?). To get the highest level of oxygen you need to a) fill the wedge with 95/5, b) get the subject to breathe this a few times to move it to the tubing, c) empty then refill the wedge, d) get the subject to hold the gas feed loosely in their hand, exhale and the inhale to fill their lungs.