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LABORATORY HANDBOOK
RESPIRATION
For the course
Human Physiology
Karolinska Institutet • Department of Physiology and Pharmacology • Physiology Education
Postal address: 171 77 Stockholm
Visiting address: Von Eulers väg 4a, plan 2
Tel exp: 08-524 872 29 • Tel vx: 08-524 864 00
RESPIRATION LABORATORY
Introduction
The main function of the lungs is gas exchange. The way in which this functions can be seen
by analysing the blood gases, i.e. O2 and CO2, of arterial blood. If one suspects that a cause of
illness is in the lungs or the chest, one can use further investigations of lung function in order
to reach a diagnosis and establish the degree of impaired function. Spirometry – measuring
lung volumes and ventilatory power – is one of these methods. During this laboratory
exercise, you will perform static and dynamic spirometry measurements. In addition, you will
investigate the acute respiratory and cardiovascular effects of hypercapnia. The purpose of
this laboratory exercise is to provide experience in two common clinical methods of
investigation and illustrate the physiological concepts within respiration.
AIMS
1. Be able to describe the components contributing to the work of breathing
2. Be aware of the major types of restrictive ventilation and which type of spirometry to use
to identify them.
3. Be able to define and measure the different lung volumes and tests of ventilation.
4. Be aware of the concept of air trapping and dynamic compression as well as understand
the flow-volume loops.
5. Be aware of the abbreviations ATPS and BTPS and know what factors are significant in
the standardization of volumes.
6. Know the effects of PCO2 and PO2 on ventilation.
7. Know the symptoms of carbon dioxide and oxygen poisoning.
8. Know the effect of breathing patterns on the size of the alveolar ventilation and by this
means PCO2 and PO2.
9. Know the effects of pronounced hyperventilation and how it is treated.
10. Know the gas exchange mechanisms of the body.
This laboratory exercise consists of three parts:
1. Static spirometry
Assessment of lung volumes
2. Dynamic spirometry
Assessment of active ventilatory power
3. Carbon dioxide re-breathing
The effects of carbon dioxide on regulation of breathing
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Human Physiology
BACKGROUND
Static spirometry
Static spirometry is used to assess lung volumes by recording inhalation- and exhalation
volumes. With the spirogram that is obtained during static spirometry, the following lung
volumes can be obtained.
IRV
TV
VC
ERV
Figure 1. Spirogram showing the variations in lung volume during normal breathing and during
maximal in- and exhalation.
Tidal volume (TV): volume of air during in- and exhalation under normal respiration
Inspiratory reserve volume (IRV): maximum volume that can be inspired following a
normal inspiration.
Expiratory reserve volume (ERV): the maximal volume that can be exhaled after a normal
expiration.
Residual Volume (RV): The volume remaining in the lung after a maximal exhalation.
Approximately 20% of the total lung capacity always remains.
Vital Capacity (VC): The maximal volume exhaled after a maximal inhalation.
(TV+IRV+ERV)
Total Lung Capacity is the volume in the lungs after a maximal inspiration (VC + RV)
Residual Quotient is RV/TLC usually about 20%.
Functional Residual Capacity (FRC) is the volume that remains in the lungs after a
normal expiration (FRC = ERV + RV).
NOTE: The sum of two or more volumes is always referred to as capacity.
The lung volumes that can be assessed during static spirometry are tidal volume,
inspiratory- and expiratory reserve volume, and the vital capacity. To measure residual
volume, a gas mixture with helium is used. Since helium diffuses slowly, it will not take
place in the gas exchange. A known volume of gas (V0), with a known helium
concentration (C0), is allowed to equilibrate with the air that remains in the lungs after a
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normal exhalation. The amount of helium is a constant. By measuring the concentration of
helium, when equilibrium is reached (C1), FRC can be measured (see fig. 4.2).
Normal range of values:
Female: VC = 4.36 x height (m) - 0.024 x age in years – 2.54. Lower limit VC – 0.9.
Male: VC = 5.19 x height (m) - 0.022 x age in years –3.03. Lower limit VC – 1.1.
Vtot=V0+FRC
V0 x C0 = Vtot x C1
Figure 2.
Vtot = (V0 x C0)/C1
V0C0
FRC
V0 +FRC = (V0 x C0)/C1
Before equilibrium
C1
C1
After equilibrium
Heliumdilution method. The person breathes in a closed system, to determine FRC and RV. A gas
mixture, with the inert gas helium, is used.
Certain diseases of the lung can give characteristic changes in the spirogram. Static
spirometry can be used in the diagnosis of restrictive lung diseases, which decreases the
normal expansion of the lung. A restrictive condition results in a decreased tidal volume,
increased breathing frequency and an increased work of breathing. The following
conditions are examples of restrictive conditions:

Reduced mobility of the thorax (kyphoscoliosis, post-operative pain, extreme obesity)

Reduced movement of the diaphragm (during pregnancy, ascites)

Decreased compliance (lung fibrosis, pneumothorax, large volume of blood in the
lungs as a result of left cardiac failure)

Reduced functional volume (tuberculosis, lung cancer).
Gas content of air
In a volume of gas of known composition at a given pressure, each gas exerts a partial
pressure, which is proportional to its share of the volume. For example, at standard
atmospheric pressure (760 mmHg) oxygen makes up 21% of the air. Therefore, at sea
level, the partial pressure for oxygen is 0.21 x 760 which corresponds to a partial pressure
of about 160 mm Hg. At a high altitude there is still 21% oxygen in the air, but since the
total pressure decreases, the partial pressure of oxygen will be reduced. This will decrease
the gas exchange resulting in an increase in ventilation.
In the table below, the partial pressures for the components of air are shown in kPa or in
parentheses as mm Hg. Although the SI notation for pressure is kPa, many physiologists
continue to use mmHg as the unit of measure for partial pressure. To convert mmHg to
kPa, divide by 7.5.
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Partial Pressure, kPa (mmHg)
Gas
Inspired air
Expired air
Alveolar air
Oxygen
21.17 (158.8)
15.33 (115)
13.33 (100)
Carbon dioxide
0.03
4.4 (33)
5.3 (40)
Water vapour
-
6.27 (47)
6.27 (47)
Nitrogen
80.13 (601)
75.33 (565)
76.4 (573)
101.3 (760)
101.3 (760)
101.3 (760)
Total pressure
Tabel 1. Partial pressure in kPa (mmHg).
As the air passes the nose and airways (dead space) it is heated to 37 oC causing water
vapour to evaporate to the inhaled air from the epithelium in the airways. As can be seen
in Table 1, the expired air is fully saturated with water vapour from the walls of the
airways (the important function of the dead space). The water vapour content of the air
depends upon the temperature and pressure (see the table on the next page). The expired
air with a temperature of 37 ºC has a Pwater of 6.27 kPa (47 mm Hg) which means that the
water content is 6.27/101.3= 6%. At 20 ºC, the air has a water content of only 2%, and the
partial pressure is thus 2.3 kPa (17.5 mm Hg).
Properties of gases and the calculation of spirometry results
As is well known, the volume of gases varies with pressure and temperature. In order to
measure the lung volumes, we breathe into a spirometer or pneumotachograph. The
volume measured must then be corrected for the temperature difference between the lungs
and the measuring apparatus as well as for the difference in water vapour content. In this
way, one obtains a standardised measurement that is not dependent on the actual
temperature.
The ideal gas law states that PV = nRT where
P is pressure
V is the gas volume
n is the number of moles
R is the gas constant (= 8.33 J x mol-2 x K-1)
T is absolute temperature in kelvins (= ºC + 273)
Lung
P0V0
/T0
BTPS
Spirometer
P0=760-47 (dry gas)
P1V1/
T1
P1=760-PH2O at room temp (rt)
V0=lung volume
V1=measured volyme
ATPS
For a quantity of gas, PV/T = constant.
V0 (BTPS) = V1 (ATPS) x ((760- Pwater)/760-47) x (310/273 + room temperature)
i.e. V0 (BTPS) = V1 (ATPS) x factor
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BTPS = body temperature and pressure, saturated
ATPS = ambient temperature and pressure
The volume in BTPS can be calculated according to the formula above or more simply by
extracting the appropriate factor from the table below.
Note: The computer will correct for this, but it is good to understand the reasoning behind
the correction.
Factors to Convert Gas Volumes from
Room Temperature Saturated to 37oC. Saturated
Factor to Convert
Volume to 37oC
When Gas
Temperature is oC
With Water
Vapor Pressure
(mm Hg)* of
1.102
1.096
1.091
1.085
1.080
1.075
1.068
1.063
1.057
1.051
1.045
1.039
1.032
1.026
1.020
1.014
1.007
1.000
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
17.5
18.7
19.8
21.1
22.4
23.8
25.2
26.7
28.3
30.0
31.8
33.7
35.7
37.7
39.9
42.2
44.6
47.0
*H2O vapour pressures from Handbook of Chemistry and Physics (34th ed.
Cleveland: Chemical Rubber Publishing Co 1952), p. 1981.
Note: These factors have been calculated for barometric pressure of 760 mmHg. Since factors at 22oC for example are
1.0904, 1.0910 and 1.0915, respectively, at barometric pressures 770, 760 and 750 mmHg. It is unnecessary to correct
for small deviations from standard barometric pressure.
The air in the lungs has a volume about 10% greater than that measured at 20 ºC.
Different gases have dissolve differently depending on the fluid present. The solubility is
proportional to the concentration in the surrounding gas phase, which is why the amount
of oxygen and carbon dioxide is dependent on their partial pressure in the alveoli. The
amount of oxygen physically dissolved in the arterial blood is quantitatively very little but
even the saturation of haemoglobin is governed by the partial pressure of oxygen which
becomes very important in situations where the partial pressure of oxygen is low such as
high altitude.
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Dynamic spirometry
Dynamic spirometry is used to measure flow, especially during expiration.
To create a flow of air in to the lungs, the surrounding tissues need to create and change
the pressure surrounding the lungs. These pressures are relatively small and are usually
described in cmH2O. The air pressure is set at zero and the other pressures are described
as deviations from this. After a normal exhalation, a negative pressure will be created by
the elastic properties of the lung and thorax. The lung tissue wants to collapse (because of
elastic fibers and surface tension) and the elasticity of the chest causes it to expand. The
difference in pressure between the alveoli and that in the pleural cavity is the
transpulmonary pressure. During an inhalation both the elastic resistance and the
resistance caused by the friction of air against the airway must be overcome. This is
increased in obstructive lungdisease. There is also friction between the thorax and lung.
Inflation of the lung is an active process that is initiated by a contraction of the
diaphragm. The chest will expand and the pleural pressure will become more negative.
The transpulmonary pressure will increase, the alveolar pressure drops below the
atmospheric pressure and air can enter the lungs.
A normal exhalation occurs due to relaxation of the muscles causing inhalation. The
volume of the thorax decreases, the pleural pressure becomes more positive, the
transpulmonary pressure decreases and the lung tissue collapses slightly due to its
elasticity and surface tension in the alveoli.
During a forced exhalation, the conditions are somewhat altered. The abdominal
musculature is activated to empty the lungs from air, and the pleural pressure will during
this type of exhalation become positive. This is due to the chest being able to reduce the
volume of the thorax faster than the lung itself can collapse. A strongly forced exhalation
will therefore not empty the lung of more air, a phenomenon called dynamic airway
compression. If the pressure in the thorax rises above that in the airway, it will be
compressed. Due to the resistance from friction, the driving force in the airway will
decrease towards the mouth. The point where the pressures inside and outside the airway
are equal is called the Equal Pressure Point (EPP). Normally, EPP will be located in the
parts of the bronchial tree where cartilage prevents compression. However, during
conditions with decreased elasticity of the lung, such as emphysema, the driving force of
the air in the airway is missing and EPP is moved towards the alveoli. When there is no
cartilaginous structures, the bronchiole will be compressed and air, peripheral to the
compression, will be trapped – air trapping.
During an obstructive disease, such as asthma, the airway friction is increased. The
airways increase in diameter during inhalations and decrease during exhalations,
especially when they are forced, due to the pressure variations in the lung that occur
during breathing. Therefore, an increase in airway friction is especially clear during a
forced exhalation. Patients with obstructive conditions will have a longer exhalation,
which may be accompanied by a wheezing. Characteristic findings with an obstructive
condition are a normal VC, but decreased FEV1.0 and FEV1.0%.
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Flows and volumes
FVC:
forced vital capacity
FEV1.0:
the volume of air forcibly expired in one second.
FEV1.0%:
FEV1.0/FVC, considered normal if above 80%
PEF:
Peak expiratory flow measured in l/min. This differs greatly between
individuals but is useful for monitoring an individual.
FEF:
Forced expiratory flow, describes the flow at a time when a given
proportion of the FVC has been expired and this expressed as FEF75 for
example. This measurement is considered to detect obstructive limitations
at an early stage, since minor limitations will be seen late in expiration.
MVV:
Maximal voluntary ventilation is the maximal volume that be breathed in
and out during a given time. Typically, it is measured over a 15 second
period and converted to l/min. MVV is reduced in both restrictive and
obstructive disease. The measurement of dynamic lung function cannot be
considered reliable in patients who are not maximally motivated during the
investigation.
Figure 3
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Carbon dioxide re-breathing: Control of respiration
Gas exchange in the body
A normal breath consists of 78% nitrogen, 21% oxygen, 1% noble gases, eventual water
vapour and a very small amount of carbon dioxide, 0.3%. (Figure 4).
Oxygen
Oxygen
Noble gas
Noble gas
Carbon dioxide
Carbon dioxide
Nitrogen
Nitrogen
Figure 4b. Expired (dry) air
Figure 4a. Inspired (dry) air
The interesting physiological question is what happens to the inspired air. A resting breath
has a volume of about 500 ml which makes up only a small part of the volume (roughly 3
l) of the ventilated parts of the lung. Therefore, the gas mixture in the alveoli does not
change much between inspiration and expiration. Oxygen diffuses into the blood and the
carbon dioxide produced by metabolism goes in the opposite direction. So when we
exhale, the composition of the expired air gives a measure of the alveolar air mixed with
dead space.
The carbon dioxide content of the expired air at the end of each breath (the end-tidal
volume) usually provides a good measure of the arterial PCO2 (about 5.3 kPa) as carbon
dioxide diffuses easily. The expired air does not provide a reliable estimate of the oxygen
content of arterial blood as oxygen does not diffuse as readily as carbon dioxide. A few
percent of carbon dioxide has been added to the expired air and about the same amount of
oxygen has been consumed. This is used to express the Respiratory Quotient (RQ);
RQ = Carbon dioxide produced/Oxygen consumed (about 0.82 at rest)
One can easily measure carbon dioxide production and thus oxygen consumption. For
example if the expired air contains 4.3% CO2, and the breathing rate is 12/min, then
Carbon dioxide production = 500 x 12/0.043 = 258 ml/min
Oxygen consumption is 258/0.82  315 ml/min.
Normal oxygen consumption is about 250 ml/min and the difference between inspired and
expired air is only a few percent. The body can easily extract the amount of oxygen that is
needed at rest, during exercise and even in the situation of heart-lung resuscitation. The
size of the alveolar ventilation depends upon amongst other things, the carbon dioxide
production and the acid-base balance.
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Procedure
Static spirometry
1. The spirometry settings of lab
chart will be open when you
start. Please do not close down
the window during the lab! You
will see two channels. Channel 1
records flow and channel 2
records changes in volume
during the breathing cycles.
Figure 5
2. Before you start the experiment, leave the mouthpiece on the table. Under the Flödemenu, in the column to the right, press Spirometer (fig. 6). The apparatus tends to
record some activity even at rest (when there is no flow). This is called drift. Correct
for this drift by pressing the Zero-button (fig 7). This assures that no signal is recorded
when the flow is zero. Wait until the computer has completed the process. Press OK.
Figure 7
Figure 6
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3. The subject, who should wear a nose-clip so that all air goes through the mouth,
should sit/ stand so that he/ she cannot see the screen. Hold the mouthpiece so that the
plastic tubes are upwards. Try breathing in the mouthpiece a couple of times before
the experiment.
4. Start the experiment by pressing the Start-button in the lower right corner. The test
will change to Stop. Breathe normally for a couple of breaths, then perform a maximal
inhalation followed directly by a maximal exhalation. Note: it does not have to be fast.
Take a couple of more breaths and finish by pressing the Stop-button.
5. To make the analysis, mark the volume-curve in channel 2 so that at least one normal
and the maximal breath are included. Among the functions in the upper toolbar, there
is a Zoom-button. Press this. Measure the lung volumes by using the marker (M) in
the lower left corner. Put the marker at a point on the curve. The volume between the
marker and the movable cross will be measured. See ”Using the computer software” if
a problem occurs. The value will be displayed as t (measured between the marker
and cross) in the textline at the top of the picture.
6. Press the button ”Chart Window” to return to the starting page (this is the case for the
whole experiment).
Zoom
Chart Window
Volumes
Value when seated (l)
Tidal volume (l)
Inspiratory reserve volume (l)
Expiratory reserve volume (l)
Vital capacity (l)
Vital capacity when laying down (l):_______________
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Check with the instructor that you have performed the measurements correctly before you
continue with the experiment.
7. When the first subject has performed the measurement, he/ she will lay down while
the other people in the group measure their lung volumes. The measurement will then,
for one of the persons in the group, be performed while lying down.
8. The other people in the group perform the measurement. For every new trial, it is
enough to press the Start-button and for analysis, mark the part of the recording that is
of importance (repeat number 3-5).
Was there a difference in vital capacity between standing and laying down?___________
What is the reason?
_______________________
_______________________
_______________________
_______________________
Note that the variation in lung volumes, even between individuals of similar size and
gender, is large, whereas a value is considered pathological only when it deviates with
more than 20% from the norm. In one and the same individual however, the values only
differ with  200 ml.
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Dynamic Spirometry
The same starting page as with the static spirometry is used. The zeroing process should
not have to be repeated.
1. Start the measurement by pressing the Start-button. The subject should breathe
normally in the mouthpiece, perform a maximal inhalation and then exhale as quickly,
as much and for as long as possible. Continue by breathing through the mouthpiece
for a while and then stop the recording by pressing the Stop-button.
2. Select this trial in both recordings for the analysis (First select one recording, then
press the [Shift]-button and hold it down as the other recoding is selected) and press
the Zoom-button. In this trial, you also use the marker and cross to find the following
parameters.
Volume and Flow
Value
Forced vital capacity (l)
Forced expiratory volume in 1 sec (l)
FEV1.0% = FEV1.0/FVC x 100 (%)
Peak expiratory flow (l/min)
3. Compare the values that you get with the values from those that the computer yields
by, when the curves are still selected, go under the Spirometry-menu and choose
Report.
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Figure 9
Carbon dioxide re-breathing
One subject will, wearing a noseclip, breathe in a closed system (bag) which from the
beginning is filled with approximately 10 l of oxygen, which will be more than the
requirement during the experiment. The trial will last for around 7 minutes, but will of
course be cancelled if the subject wishes to do so. During the experiment, carbon dioxide
from the subject’s exhaled air, will be collected in the bag and in the subject’s tissues.
A small flow from the mouthpiece is pumped through a gas analyzer, which analyzes the
levels of CO2 and O2. The expired flow will also pass through a pneumotachometer where
flow, breathing frequency and tidal volume is recorded.
Heart rate will be measured with a heart rate monitor and blood pressure will be measured
with auscultation.
You will need the following for the experiment:







Subject.
Record keeper. The results will be registered on a whiteboard every minute.
Someone who takes blood pressure.
One person who asks for symptoms according to the protocol. The subject will show
the number of fingers that represent the degree of difficulty (1 is the least and 5 is the
most difficult).
Timer who informs the other group members about the time every minute.
Someone to who reads the values off the computer.
One person who reads the heart rate off the heart rate monitor.
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1. Divide the task among the group members.
2. The subject puts on the heart rate monitor. The strap should be moistened and placed
around the chest. Place the blood pressure cuff on the subjects right arm.
3. Get resting values. Press START. Record for 2-3 min. The subject should breathe as
normally as possible in the mouthpiece, which should not be attached to the bag. Press
STOP. Measure blood pressure. Record the resting values.
4. The instructor will fill the bag with 100% oxygen and connect it to the mouthpiece.
The subject will re-breathe through the bag. All measurements and questions will be
repeated every minute.
5. When the subject aborts or when the endtidal CO2 reaches 7%, the mouthpiece is
disconnected from the bag and the subject breathes air through the mouthpiece. The
experiment ends when values return to normal.
Rest
1 min
2 min
3 min
4 min
5 min
6 min
7 min
8 min
Breathing freq.
(b/min)
Tidal volume
(l)
Flow (l/sec)
Endtidal
% CO2
Heartrate
b/min)
Blood pressure
(mmHg)
Dyspnea
Headache
Warmth
6. The subject describes what the experiment felt like!
7. How and why do the parameters in the protocol change when the subject breathes in
the bag? Discuss and ask the instructor for help.
8. Analyze the CO2-curve. Can you read the partial pressure of CO2 in the subject’s
arterial blood? PO2?
9. Which gas, CO2 or O2 in arterial blood regulates breathing in healthy individuals
under normal conditions?
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