Week 4 - week 4 Prac PDF

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La Trobe University

Dept of Physiology, Anatomy & Microbiology

Week 4 - Wednesday Analysis of Expired Air OBJECTIVES

Gas volume data in respiratory physiology are presented in one of three forms:

To measure the composition of gases in the mixed expired air.

(a) VATPS (Volume at Ambient Temperature and Pressure, Saturated); as it was measured under laboratory conditions i.e., at room temperature and pressure, and with a temperature-dependent water vapour pressure.

To calculate the volume of the respiratory dead space and the alveolar ventilation. To calculate oxygen consumption and carbon dioxide production from these data.

(b) VSTPD (Volume at Standard Temperature and Pressure, Dry); converted from VATPS to standard temperature and pressure conditions as a dry gas. By universal convention, V󰇗 O2 and V󰇗 CO2 are expressed as STPD, as this permits comparisons of values to be made independently of the altitude of the laboratory where the measurements were made. (c) VBTPS (Volume at Body Temperature and Pressure, Saturated); converted from laboratory conditions to physiological conditions. The gas volumes measured in the laboratory (i.e., VATPS) are converted to the conditions in the lungs, i.e., body temperature and pressure, saturated with water vapour at 37°C. The point of this volume transformation is as follows: air is exhaled from the lungs "warm and wet" (BTPS), but is measured "cold and dry" (ATPS). We wish to know the value for tidal volume, minute ventilation, alveolar ventilation, under physiological conditions (37°C, PH2O = 47 mmHg) rather than under laboratory conditions (e.g. 22°C, PH2O = 19.8 mmHg). The volume measured will be less than the volume expired due to the effect of cooling; secondly condensation of water vapour may change the measured volume below that actually exhaled from the subject, dependent again on ambient temperature. In this Practical class, expired gas will be collected and analysed at ATPS, and converted into the appropriate form for presentation of lung volumes and gas exchange rates.

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

EXPERIMENTAL PROCEDURE Measurements of many respiratory parameters are not difficult to make, as you will find in this series of experiments. A timed collection of expired air into a Douglas Bag is used to calculate all of the respiratory variables in this laboratory class. Despite the apparent simplicity of this task, accuracy of measurement is important both in determining lung volumes and in gas analysis. Small errors in measurement may lead to large errors in the final result and to possible misinterpretation of clinical data. The experiments will also show the importance of the subject being in a quiet resting state since there are multiple inputs to the respiratory centres from other regions of the CNS. valve block

face mask

tubing Douglas bag

tap

Figure 1: The components of the Douglas Bag

sampling tube (with screw clip)

As shown in Figure 1, a tap is fitted to the Douglas bag. It is important that the tap is fitted in the right way: the tap must be fitted to permit the subject's expired gas to pass either back into the room, or into the bag. Next comes a flexible hose to which is fitted a two-way valve with an attached face mask. Before attaching the valve block to the hose pipe, fit the face-mask to the central port and inhale and exhale while checking with a spare hand the direction of flow through the valve block. The outlet of the valve block is inserted into the hose pipe. The subject should be able to inhale easily and expire into the hose. Prior to collection of gases, ensure that the side sampling tube of the bag is blocked off by the screw clip.

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

ACTIVITY ONE – Collection of Expired Air 1. The subject should be in a seated, rested position and breathe into the Douglas bag with the facemask attached (please note that the facemask adds an additional ~150ml to the subject’s dead space volume). 2. Before beginning the activity, the bag and the small sampling tube must be rinsed out with the subject's expired gases to displace any residual gas in the bag. The subject breathes into the bag for a few minutes with the side arm clamped. Then the bag is completely emptied of expired air by rolling it up such that the air is pushed out of the tap. In order to also rinse the sampling tube, it is important that the side tube is unclamped during this procedure. Be firm but gentle when squeezing the bag to completely empty it. This will ensure that an inevitable dead space of the bag is filled with expired air. Once the bag has been emptied, close the tap and re-clamp the side tube. 3. The subject is now re-attached to the bag and the tap is set so that the expired gases are not collected, but are returned to the room. When the subject's breathing returns towards a normal involuntary pattern (usually you need to wait at least 5 min), proceed with the experimental collection. Do not be tempted to cut short this period - good results depend on your subject breathing as normally as possible. 4. When breathing is relaxed and regular turn the tap to direct the subject's expired gases into the bag and commence collecting gases. Allow the subject to breath into the bag for 5-7 min, making sure that one member of the team counts the number of breaths taken by the subject during the collection period (note that it does not really matter exactly how long the collection period is, as long as the length of the collection period and the number of breaths taken by the subject are known accurately). 5. After ~5 min you should have collected enough expired gas to noticeably inflate the bag. Close the tap to the bag and continue with your analysis.

(A) Expired Gas Concentration: 1. The PowerLab gas analyser used to measure the percentage of O2 and CO2 in gas samples should be switched “on” and the “record” button clicked. Please ensure that the pump is switched to ‘ON’ so that air will be drawn into the analyser. 2. Once the chart commences recording ensure the readout indicates ~21.0% O2 and ~0.03% CO2 when room air is drawn through the sample tube (the machine will have been calibrated previously by the laboratory staff using a gas mixture of exact composition). 3. Air is drawn into the analyser by the pump. Unclamp the side tube of the bag, connect the sample tube to it, and allow gas to be drawn through the analyser for ~30s. Do not force air out of the bag or erroneous readings will result AND you may damage the analyser. The flow rate of the pump is displayed on top of the machine – by knowing this flow rate, and the time that you have 3

HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

been sampling (~30s) you will be able to calculate the volume of gas drawn from the bag by the gas analyser (the sampled volume must be added to the total volume in the bag which you will determine next). 4. Disconnect the sample tube and re-clamp the side tube on the bag.

(B) Expired Gas Volume: The volume of expired gas in the bag is measured using the Aichi-Tokei gas meter. The dial reading is a cumulative digital measure of volume, and cannot be reset to zero. The right-most digit is litres, and this is further divided into 200 ml units by graduations to the right of this digit. 1. Check the direction of gas flow (arrow on the top of the meter) and connect the hose pipe from the Douglas bag to the inlet of the meter. Note the initial meter reading. Do not waste time setting the initial reading to a convenient "round" value. 2. Turn on the tap and roll out the Douglas bag. If the bag requires re-rolling to exhaust the gas, turn off the tap and rearrange the bag before re-rolling the bag. 3. Take the final reading when the bag is exhausted, and subtract the initial reading (the equation below will assist you). Enter your data into the Table below VE (ATPS)

= (Final value - Initial value) + volume sampled with analyser = …………….… L (ATPS) Collection Period (min) Douglas Bag

Total Breaths Respiratory rate (per min) Room air %O2 Douglas Bag

Expired Gas Concentration

Room air %CO2 Douglas Bag Volume sampled (ATPS)

Expired Gas Volume

Total Volume of Douglas Bag(L; ATPS)

Table 1: Analysis of Expired Air

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

ACTIVITY TWO – Calculating the Conversion Factors The values for room temperature (°C) and atmospheric pressure (PB) will be measured by a demonstrator and written on the whiteboard. Water vapour pressure (PH2O) is the saturated vapour pressure of water at the ambient temperature T°C and can be found in the table below. ToC

PH2O

ToC

PH2O

ToC

PH2O

ToC

PH2O

ToC

PH2O

15

12.8

20

17.5

25

23.7

30

31.8

35

42.1

16

13.7

21

18.6

26

25.2

31

33.7

36

44.5

17

14.5

22

19.8

27

26.7

32

35.6

37

47.0

18

15.5

23

21.1

28

28.3

33

37.7

38

49.7

19

16.4

24

22.4

29

30.0

34

39.9

39

52.4

Calculate the conversion factors for the following transformations: To convert VATPS to VBTPS:



VBTPS

310 273 + To C

= VATPS x [

×

PB − PH2O PB − 47

]

= VATPS x [........................]

 To convert VATPS to VSTPD: VSTPD

= VATPS x [

273

273 + To C

×

PB − PH2O 760

]

= VATPS x [........................]



To convert VBTPS to VSTPD: VSTPD

PB − 47 273 ] × 760 310

= VBTPS x [

= VBTPS x [........................]



To convert VSTPD to VBTPS: VBTPS

760 310 ] × PB − 47 273

= VSTPD x [

= VSTPD x [........................]

These conversion factors will be needed in the following calculations (Activity Three). 5

HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

ACTIVITY THREE – Calculations from Expired Air Please note: V = volume of gas, while V󰇗 (note the dot above the V) = volume of gas flow per unit time (i) Respiratory Minute Volume (𝐕󰇗 E): V󰇗E =

V

and;

time

VT =

V󰇗E

resp. rate

This may be adjusted to both STPD and BTPS by using the conversion factors that you calculated in Activity Two (subscript E = expired). Complete Table 2. V󰇗 E (L/min)

VT (L)

ATPS BTPS STPD Table 2: Respiratory minute volume

(ii) Dead Space Volume (VD): Dead space volume is calculated from the Bohr equation shown below. Note: V󰇗D is expressed under BTPS. V󰇗D V󰇗E

Where:

=

(FA CO2 − FE CO2 ) FA CO2

𝑉󰇗𝐸 (BTPS) = .......... (calculated above) FACO2 = 0.056 (alveolar gas is 5.6% CO2 at sea level) FECO2 = .......... (measured as % in Table 1 but then converted to a fraction) V󰇗D = .......... L/min (BTPS) (enter this value in Table 3) VD = .......... L (BTPS) (enter this value in Table 3)

(iii) Alveolar ventilation (𝐕󰇗 A): This is the difference between V󰇗 E and V󰇗 D, both expressed under BTPS:

V󰇗A = V󰇗E - V󰇗D = ............. L/min (BTPS) - ............. L/min (BTPS) = ............. L/min (BTPS) (enter this value in Table 3)

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

Please note: V󰇗 A will later be needed in the units STPD so convert below: V󰇗 A (STPD)

= V󰇗 A L/min (BTPS) x conversion factor (STPD from BTPS) = .................... L/min (BTPS) x …………………. (from Activity Two) = ............... L/min (STPD) (enter this value in Table 3)

(iv) O2 utilized in 1 minute (𝐕󰇗 O2): The volume of O2 diffusing across the lungs per minute is the difference between the amount entering the alveoli during inspiration and the amount leaving the alveoli during expiration. Note that in the following equation, V󰇗 A is used rather than V󰇗 E, since V󰇗 E includes a large percentage of ‘fresh’ air from the dead space. Also note that you need to use fractions of O2 rather than percentages: V󰇗O2 = (FIO2 x V󰇗A) - (FAO2 x V󰇗A) Where:

FIO2 = fraction of O2 in inspired (= room) air (see Table 1) 𝑉󰇗 A (STPD) = alveolar ventilation, see Table 3 FAO2 = fraction of O2 in alveolar air (= 0.14) V󰇗 O2 = (............ x ............ ) - (0.14 x ........... ) = ............ L/min (STPD) (enter this value in Table 3)

(v) CO2 produced in 1 minute (𝐕󰇗 CO2): Using the same principle as stated above: V󰇗CO2 = (FACO2 x V󰇗A) - (FICO2 x V󰇗A) Since FICO2 (fraction of CO2 in room air) is essentially zero, the equation can be simplified to: V󰇗 CO2 = FACO2 x V󰇗 A where:

FACO2 = 0.056 V󰇗 A (STPD) = alveolar ventilation, see Table 3 V󰇗 CO2 = .................. L/min (STPD) (enter this value in Table 3)

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

Variable

Units

V󰇗 E (L/min)

BTPS

VT (L)

BTPS

VD (ml)

BTPS

V󰇗 D (L/min)

BTPS

V󰇗 A (L/min)

BTPS

V󰇗 A (L/min)

STPD

V󰇗 O2 (L/min)

STPD

V󰇗 CO2 (L/min)

STPD

Value

Table 3: Summary of calculated values in the accepted units

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HBS2PPB Human Physiology Practicum B

Lab notes 2018

La Trobe University

Dept of Physiology, Anatomy & Microbiology

Week 4 - Thursday Analysis of Expired Air OBJECTIVES To clarify any concepts/technical details from Wednesday’s prac as necessary To complete the weekly MCQ

In today’s workshop your demonstrators will take you through yesterday’s prac, concentrating on the major concepts that were covered and giving you an opportunity to seek any feedback/clarification before you complete the weekly quiz. You will also have the opportunity to clarify any questions you have about this week’s assigned reading. Please make sure you bring a copy of the paper as well as this week’s lab notes to the workshop.

Submission for today’s session: The last 20 min of today’s workshop will consist of a quiz which you will complete individually under open-book conditions (i.e. you will be allowed to refer to notes and/or this week’s paper, but NO INTERNET RESOURCES CAN BE USED). The quiz will test your understanding of this week’s prac and also your ability to read and comprehend this week’s scientific manuscript. Good luck!

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HBS2PPB Human Physiology Practicum B

Lab notes 2018...