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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 • Fax: 08-524 872 31 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 that make up the laboratory 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 Respiration - HLR 2 OSF, T3, Läkarprogrammet 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 = IRV X 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 is 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 Human Physiology Respiration 1 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 C1 V0 +FRC = (V0 x C0)/C1 Before equilibrium C1 After equilibrium Heliumdilution method. The person breaths 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 is 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 mm Hg) 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 Human Physiology Respiration 2 continue to use mm Hg as the unit of measure for partial pressure. To convert mm Hg to kPa, divide by 7.5. Partial Pressure, kPa (mm Hg) 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 37oC causing water vapour to evaporate to the inhaled air from the epithelium in the airways. As can be seen, 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 know, 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 correct 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. Human Physiology Respiration 3 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 Spirometer P0=760-47 (dry gas) P0V0 /T0 P1V1/ T1 P1=760-PH2O at room temp (rt) V0=lung volume V1=measured volyme BTPS 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 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. Human Physiology Respiration 4 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. Human Physiology Respiration 5 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 an 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, Human Physiology Respiration 6 which may be accompanied by a wheezing. Characteristic findings with an obstructive condition is a normal VC, but decreased FEV1.0 and FEV1.0% . 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 Human Physiology Respiration 7 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. Human Physiology Respiration 8 Procedure Static spirometry 1. Double-click the Spirometriikon in the Respiration-folder on the desktop. You will see two channels. Channel 1 records flow and channel 2 records changes in volume during the breathing cycles. Maximize the size of the window. 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 6 Figure 7 Human Physiology Respiration 9 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 is 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 Δt (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):_______________ Human Physiology Respiration 10 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 laying 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. Human Physiology Respiration 11 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. Human Physiology Respiration Figure 8 12 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. Human Physiology Respiration 13 1. Divide the task among the group members. Double-click the CO2-återandning icon in the Respiration folder. 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. Zero the spirometer according to number 2 under static spirometry. 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 wil 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 norma. 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 Effort 6. The subject describes what the experiment felt like! Human Physiology Respiration 14 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 healty individuals under normal conditions? Human Physiology Respiration 15