Recording established using the VO2 and RER graph template file (h19.gtl)

Updated 10-14-13 BSL PRO Lesson H19: VO 2 and RER Measurement Recording established using the VO2 and RER graph template file (h19.gtl) Overview Oxygen Consumption (VO 2 ) and Respiratory Exchange Ratio (RER) Real-time Oxygen Consumption (VO 2 ) and Respiratory Exchange Ratio (RER) measurements can be obtained using the MP3x System with the Gas-System2 module and some airflow accessories. When performing tests of this kind, many factors exist to confound the measurement. For example, it may be reasonable to think that the volume of expired air is always the same as inspired air, but this is only true when the volume of carbon dioxide expired is equal to the volume of oxygen consumed. The inspired and expired volumes are equal only when the RER equals one. RER is defined as VCO 2 produced divided by VO 2 (where V is volume). Accordingly, when performing these measurements, precise determination of inspired and expired volumes and accurate assessment of the gas concentration level are required. The Gas-System2 measures O 2 and CO 2 concentrations in vapor. When the subject inspires, air will be drawn into the Gas-System2 through the SS11LA airflow transducer. The SS11LA is placed on the inspiration side to eliminate any effects associated with www.biopac.com Page 1 of 16

expired air humidity (see Hardware Setup for more detail). When the subject expires, air will be directed to the Gas-system2 module. The Gas-System2 is designed to work with saturated expired air. When using a mixing chamber to average O 2 and CO 2 concentrations over several breaths and measuring these changes for arbitrarily high breathing rates, there is no performance degradation. Accordingly, a mixing chamber (such as the Gas-System2 utilizes) is typically recommended for quick, accurate and easy metabolic analysis. IMPORTANT CONCEPT! The non-rebreathing T valve directs only expired air to the Gas-System2. Because only expired air is directed to the module, the system acts to average respiratory outflows. This averaging effect causes the CO 2 and O 2 concentrations to vary in accordance to the mean values resident in a few expired breaths. The size of the system s chamber determines the extent of the averaging effect. For example, if the subject s expired breath volume is 0.5 liters, the Gas-System2 s chamber will average about 10 expired breaths. Definitions Click here for definitions of abbreviations used in this lesson and the calculation channel Expression formulas used for VO 2 and RER. Objectives 1. To obtain absolute VO 2 and RER values for a subject at rest (optional segments include hyperventilation and recovery from exercise). 2. To demonstrate the relationship between VO 2 and RER. NOTE: The Gas-System2 module is intended for VO2 and RER studies ranging from resting to moderate exercise. For prolonged or vigorous exercise studies where CO2 levels may exceed 5%, BIOPAC recommends the AcqKnowledge Research system with O2100C and CO2100C gas sensing modules. Equipment BIOPAC Gas Analysis System (GASSYS2-EA) BIOPAC Pneumotach Airflow Transducer [High Flow] (SS11LA) BIOPAC Calibration Syringe (AFT26 or AFT6) BIOPAC 22 mm Non-Rebreathing T Valve (AFT22) BIOPAC Rigid Coupler (AFT11E) for T-valve to tubing BIOPAC Coupler (AFT11C) for T-valve to calibration syringe BIOPAC Smooth Bore Tubing-35 mm (AFT7) BIOPAC Disposable Nose Clip (AFT3) BIOPAC Disposable Mouthpiece (AFT2) BIOPAC Disposable Bacterial Filters (AFT1) requires 2 filters Computer running Windows XP or higher, or Mac OS X Biopac Student Lab PRO software BIOPAC Data Acquisition Unit (MP36/MP35/MP30) OPTIONAL: BIOPAC Calibration Gas Cylinder (16% O 2, 4% CO 2 ), with regulator and tubing The following measures are also required: Ambient temperature in C Ambient barometric pressure in mmhg Pressure of water vapor at ambient temperature = 22.4 mmhg www.biopac.com Page 2 of 16

Setup Hardware The Gas-System2 sensors are factory calibrated prior to shipping. The O 2 sensor is a zirconia solid electrolyte with a 0.1-25% sensing range and an estimated 5-year lifetime. It runs hot, which helps to burn off humidity. The CO 2 sensor uses a humidity-repellant (hydroponic) membrane and has a sensing range of 0-5%. It uses non-dispersive infra-red diffusion with single-beam IR and a self-calibrating algorithm. It also runs hot, which helps to burn off humidity. 1. The Gas-System2 module is supplied with a 5 VDC @ 4 amp wall adapter. Plug the adapter into the main power supply and insert the adapter plug into the DC Input on front of the Gas-System2 module. 2. Flip up the power switch on the Gas-System2 to turn it "ON." o The unit should warm up for at least five minutes before calibration, so it is suggested that you turn it on now and then complete setup. 3. Plug the transducers into the MP3X as follows: Transducer MP3X CH # SS11LA CH 1 O2 line (from the Gas-System2) CH 2 CO2 line (from the Gas-System2) CH 3 www.biopac.com Page 3 of 16

4. Turn on the MP unit (assuming the AC100A power adapter has already been connected). 5. Connect the airflow accessories as follows: Air Flow Accessory AFT1 filter SS11LA transducer AFT22 non-rebreathing T-valve AFT11E coupler AFT7 tubing AFT26 or AFT6 calibration syringe AFT11C coupler Connects to SS11LA transducer, on the "Inlet" side AFT22 non-rebreathing T-valve AFT11E coupler AFT7 tubing Gas-System2 inlet (back side) AFT11C coupler AFT22 non-rebreathing T-valve Note: The above setup uses the AFT26 or AFT6 calibration syringe. For recording, you must replace the calibration syringe with an AFT1 filter and an AFT2 disposable mouthpiece (connected to each other and to the AFT22 T-Valve). www.biopac.com Page 4 of 16

AFT7 tubing to Gas-System2 inlet 6. Pump the calibration syringe through enough cycles to fill the chamber with ambient air. o AFT26, pump 3-4 times. o AFT6, pump 10-12 times. Software 1. Launch the BSL PRO software on the host computer. The program should create a new "Untitled1" window. 2. Open the VO 2 and RER Template by choosing File > Open > choose Files of type: Graph Template (*GTL) > File name: H19 V02 & RER.gtl The template will establish the required settings. This particular setup will provide a reading that, at any point in time, indicates the absolute volume of oxygen consumed, carbon dioxide produced, and the respective respiratory exchange ratio in the last 60 seconds. Units are liters because it is a volume measure, not a flow rate. 3. Save As the desired file name. Calibration The following calibration steps are outlined below: A. Calibrate flow transducer B. Calibrate O 2 channel (CH2) C. Calibrate CO 2 channel (CH3) D. Normalize moist gas to volume occupied by dry gas at 0 C, 760 mm. E. Optional: Calibrate for time interval (template is based on 60-second interval). Note: In the calculation channel formulas, the following assumed values were used for gas concentrations in normal atmosphere (chamber flooded with ambient air): 0 2 = 20.93% CO 2 = 0.04% N 2 = 79.03% The optional Stage 2 gas calibration is not required, but can be performed using the small line inlet on the back panel of the Gas-System2 module. If you introduce calibration gases into the chamber, adjust the calculation channel expressions for the real gas values. If you perform a gas calibration, use the following ranges: Flow (inspired): calibrate within the range of 0 to 13 liters/sec O 2 concentration: calibrate in range of 21% to 16% O 2 CO 2 concentration: calibrate in range of.04% to 4% CO 2 www.biopac.com Page 5 of 16

Flow 1. Select MP3X > Setup Channels. 2. Click the wrench icon for CH 1. 3. Click the Scaling button. 4. Hold the Calibration Syringe such that the SS11LA hangs from it in an upright position. 5. Click on the Cal1 button. 6. Subtract 3,000 from the Cal1 input value and enter the result in Cal2 input value (Cal2 input value should be 3,000 less than Cal1 input value). 7. Confirm Cal1 scale value= 0 and Cal2 scale value=10. 8. Click OK. 9. Set the equipment down. www.biopac.com Page 6 of 16

O 2 Per Setup, the chamber should have been flooded with ambient air prior to O 2 calibration! 1. Click the wrench icon for CH 2. 2. Click the Scaling button. 3. Click the Cal2 button. 4. Enter 20.93 into the Cal2 scale value. 5. Confirm both Cal1 input value= 0 and Cal1 scale value=0. 6. Click OK. CO 2 Per Setup, the chamber should have been flooded with ambient air prior to CO 2 calibration! 1. Click the wrench icon for CH 3. 2. Click the Scaling button. 3. Click the Cal1 button. 4. Enter 0.04 into the Cal1 scale value. 5. Add 10 to the Cal1 input value and enter the result as Cal2 input value. 6. Confirm Cal2 scale value=1.04 7. Click OK. Note: The sensor output is 10 mv per 1% C0 2. With proper calibration, if the sensor rails, it will read as 5% CO 2. www.biopac.com Page 7 of 16

OPTIONAL Stage 2 Calibration (BIOPAC Calibration Gas Cylinder required) O 2 Per Setup, the chamber should NOW be flooded with a calibrated gas (16% O 2, 4% CO 2 )! 1. Click the wrench icon for CH 2. 2. Click the Scaling button. 3. Click the Cal1 button. 4. Enter 16 into the Cal1 scale value. 5. Click OK to dismiss scaling dialog. 6. Click OK to dismiss Input Channel Parameters dialog for CH 2. CO 2 Per Setup, the chamber should NOW be flooded with a calibrated gas (16% O 2, 4% CO 2 )! 1. Click the wrench icon for CH 3. 2. Click the Scaling button. 3. Click the Cal2 button. 4. Enter 4 into the Cal2 input value. 5. Click OK to dismiss the scaling dialog. 6. Click OK to dismiss Input Channel Parameters dialog for CH 3. www.biopac.com Page 8 of 16

Gas Normalization Normalize moist gas to volume occupied by dry gas at 0 deg C, 760mm 1. Use this table to find the normalization factor. 2. Click the wrench icon for C3 Vis (STPD). 3. Enter the normalization factor in the formula: C2*(normalization factor) Be sure to enter the value as 0.x, with the leading decimal. 4. Click OK. Optional: Interval Only required if you change the time interval from the template, which is set for a 60-second interval. 1. Click the wrench icon for C2. www.biopac.com Page 9 of 16

3. Calculate the Samples entry as follows: Integration Samples = Time Interval x Sample rate (Template setting: Integration Samples = 60 sec x 100 samples/sec = 6000 samples) 4. Enter the new Samples value. 5. Click OK. Recording This lesson records absolute volume of O 2 and CO 2 in a 60-second interval. Units are liters because this provides a volume measure, not a flow rate. If you change the time interval, you must adjust the Integrate formula. Hints for minimizing measurement error: 1. Ensure that the connections among the various pieces of equipment are fairly tight to prevent contamination via room air. 2. Try to start the recording immediately after a full exhale. This will prevent receiving results with an O 2 inspiration value less than that of the total expiration value (although the averaging will minimize the inaccuracies that this would cause). 3. It's very important that any extraneous volumes are minimized between the subject and the T-valve. Additional volumes affect the effective expired gas concentration levels. 4. The tubing must be connected to the SS11LA on the unlabeled side (that does NOT say "Inlet"). Preparation 1. Remove the calibration syringe from the transducer setup. 2. Establish the recording connections as shown below: Airflow Accessory Connects to AFT1 Filter AFT22 T-Valve (in the existing setup) AFT2 disposable mouthpiece AFT1 Filter www.biopac.com Page 10 of 16

Segment 1 1. Have the subject put a nose clip on. 2. Click START in the PRO software. 3. Have the subject breathe normally for at least 2 minutes. o Subject must breathe long enough to ensure that the 5-liter mixing tank has been filled, thereby enabling it to average those breaths. o The subject should breathe into the disposable bacterial filter (AFT1) through a disposable mouthpiece attachment (AFT2). 4. Click STOP in the PRO software. Breathe through the mouthpiece www.biopac.com Page 11 of 16

Optional Segments Recovery from Exercise 1. Have the subject perform several minutes of medium-to-strenuous exercise. o If desired, record the heart rate and correlate VO 2 to heart rate. 2. Click START in the PRO software. 3. Have the subject breathe normally for at least 2 minutes. 4. Click STOP in the PRO software. Hyperventilation 1. Click START in the PRO software. 2. Have the subject breathe normally for at least 1 minute. 3. Have the subject hyperventilate for at least 1 minute; enter a marker at start of hyperventilation (Esc on PC, F9 on Mac). 4. Have the subject breathe normally for at least 1 minute. 5. Click STOP in the PRO software. Notes To save recorded data, choose File menu > Save As > file type: BSL PRO files (*.ACQ) File name: (Enter Name) > Save button To erase all recorded data (make sure you have saved it first), and begin from Time 0, choose: MP menu > Setup Acquisition > Click Reset www.biopac.com Page 12 of 16

Analysis The VO 2 and RER template will display channels as follows: Channel Displays Measure CH 1 Fi (inspired Air Flow) Value CH 2 O 2 E (expired O 2 ) Value CH 3 CO 2 E (expired CO 2 ) Value CH 40 (C1) N 2 E (expired N 2 ) Value CH 41 (C2) Vi (ATP) Value CH 42 (C3) VIS (STPD; inspired) Value CH 43 (C4) VES (STPD; expired) Value CH 44 (C5) VO 2 (STPD) Value CH 45 (C6) VCO 2 (STPD) Value CH 46 (C7) RER Value 1. Channels 40-43 are not displayed because they are calculation channels required for conversions. You can show/hide channels by Ctrl-clicking the channel box. 2. Note that the VO 2 and RER measurements (bottom two channels) vary smoothly with time. The graphical and continuous nature of this recording and calculation method provides significant information regarding the changes of these variables over time. 3. To display measurements, click the measurement icon. 4. To display markers, click the flag icon. 5. To display grids, click the grid icon. www.biopac.com Page 13 of 16

Appendix GRAPH TEMPLATE SETTINGS Click here to open a PDF of the graph template file settings. The BSL PRO Graph Template file automatically establishes the settings shown in the table. Definitions These abbreviations are used in the discussion and calculation channel Expression formulas for VO 2 and RER: ATP Concentration of gas at ambient temperature and pressure CO 2 Fi Carbon dioxide Est. concentration ambient environment: 0.04% CO 2 e = Carbon Dioxide fractional concentration in expired air Inspired air flow (ATP) N 2 Nitrogen N 2 e = Nitrogen fractional concentration in expired air N 2 e = 100 (CO 2 e + O 2 e) O 2 Pb PH 2 0 RER STPD Oxygen Est. concentration in ambient air: 20.93% O 2 e = Oxygen fractional concentration in expired air Est. concentration in expired breath: 16% oxygen Ambient barometric pressure (e.g. 745 mmhg) Ambient pressure of water vapor (e.g. 22.4 mmhg) Respiratory Exchange Ratio RER = VCO 2 / VO 2 Concentration of gas at standard temperature and pressure, dry Ta Ambient temperature (e.g. 24 deg. C) VCO 2 VO 2 Vi Volume of carbon dioxide produced (STPD) per 60-sec interval Real-time carbon dioxide production VCO 2 = (1/100)*[(Ves* CO 2 e) (Vis*.04)] Volume of oxygen consumed (STPD) per 60-sec interval Real-time oxygen consumption VO 2 = (1/100)*[(Vis*20.93) (Ves*O 2 e)] Inspired air volume (ATP) per 60-sec interval Vi = Integrate (Fi) over 60 seconds. The number of samples is equal to the time interval x sample rate (template is 60 sec. x 100 samples/sec = 6000 samples). Select "Average over samples." Vis Inspired air volume (STPD) per 60-sec interval Vis = C2 * Normalization Factor (see Table of Normalization Factors) If normalization factors are unavailable, you can use this formula: Vis = Vi*(273/(273+Ta))*((Pb-PH 2 0)/760) where Ta = ambient temperature Pb = barometric pressure PH 2 O = 22.4 mmhg (assumed value) Ves Expired air volume (STPD) per 60-sec interval Ves = (Vis*79.03) / N 2 e Note: If you change the time interval from 60 seconds, you must adjust the Vi formula. www.biopac.com Page 14 of 16

Normalization Factors The following table lists the factors required to reduce volume of moist gas to volume occupied by dry gas at 0 C, 760 mm. OBR is an abbreviation for observed barometric reading, uncorrected for temperature. Fahrenheit values are roughly converted and rounded from Celsius values as T F =(9/5*T C )+32ºF. Important! All factors are 0.x, with x being the value from the table. Be sure to include the leading decimal when you complete your calculation. ºC 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 ºF 59 61 63 64 66 68 69 72 73 75 77 79 81 82 84 86 88 90 OBR 700 855 851 847 842 838 834 829 825 821 816 812 807 802 797 793 788 783 778 702 857 853 849 845 840 836 832 827 823 818 814 809 805 800 795 790 785 780 704 860 856 852 847 843 839 834 830 825 821 816 812 807 802 797 792 787 783 706 862 858 854 840 845 841 837 832 828 823 819 814 810 804 800 795 790 785 708 865 861 856 852 848 843 839 834 830 825 821 816 812 807 802 797 792 787 710 867 863 859 855 850 846 842 837 833 828 824 819 814 809 804 799 795 790 712 870 866 861 857 853 848 844 839 836 830 826 821 817 812 807 802 797 793 714 872 868 864 859 855 851 846 842 837 833 828 824 819 814 809 804 799 794 716 875 871 866 862 858 853 849 844 840 835 831 826 822 816 812 807 802 797 718 877 873 869 864 860 856 851 847 842 838 833 828 824 819 814 809 804 799 720 880 876 871 867 863 858 854 849 845 840 836 831 826 821 816 812 807 802 722 882 878 874 869 865 861 856 852 847 843 838 833 829 824 819 814 809 804 724 885 880 866 872 867 863 858 854 849 845 840 835 831 826 821 816 811 806 726 887 883 879 874 870 866 861 856 852 847 843 838 833 829 824 818 813 808 728 890 886 881 877 872 868 863 859 854 850 845 840 836 831 826 821 816 811 730 892 888 884 879 875 871 866 861 857 852 847 843 838 833 828 823 818 813 732 895 890 886 882 877 873 868 864 859 854 850 845 840 836 831 825 820 815 734 897 893 889 884 880 875 871 866 862 857 852 847 843 838 833 828 823 818 736 900 895 891 887 882 878 873 869 864 859 855 850 845 840 835 830 825 820 738 902 898 894 889 885 880 876 871 866 862 857 852 848 843 838 833 828 822 740 905 900 896 892 887 883 878 874 869 864 860 855 850 845 840 835 830 825 742 907 903 898 894 890 885 881 876 871 867 862 857 852 847 842 837 832 827 744 910 906 901 897 892 888 883 878 874 869 864 859 855 850 845 840 834 829 746 912 908 903 899 895 890 886 881 876 872 867 862 857 852 847 842 837 832 748 915 910 906 901 897 892 888 883 879 874 869 864 860 854 850 845 839 834 750 917 913 908 904 900 895 890 886 881 876 872 867 862 857 852 847 842 837 752 920 915 911 906 902 897 893 888 883 879 874 869 864 859 854 849 844 839 754 922 918 913 909 904 900 895 891 886 881 876 872 867 862 857 852 846 841 756 925 920 916 911 907 902 898 893 888 883 879 874 869 864 859 854 849 844 758 927 923 918 914 909 905 900 896 891 886 881 876 872 866 861 856 851 846 760 930 925 921 916 912 907 902 898 893 888 883 879 874 869 864 859 854 848 www.biopac.com Page 15 of 16

762 932 928 923 919 914 910 905 900 896 891 886 881 876 871 866 861 856 851 764 936 930 926 921 916 912 907 903 898 893 888 884 879 874 869 864 858 853 766 937 933 928 924 919 915 910 905 900 896 891 886 881 876 871 866 861 855 768 940 935 931 926 922 917 912 908 903 898 893 888 883 878 873 868 863 858 770 942 938 933 928 924 919 915 910 905 901 896 891 886 881 876 871 865 860 772 945 940 936 931 926 922 917 912 908 903 898 893 888 883 878 873 868 862 774 947 943 938 933 929 924 920 915 910 905 901 896 891 886 880 875 870 865 776 950 945 941 936 931 927 922 917 912 908 903 898 893 888 883 878 872 867 778 952 948 943 938 934 929 924 920 915 910 905 900 895 890 885 880 875 869 780 955 950 945 941 936 932 927 922 917 912 908 903 898 892 887 882 877 872 ºF 59 61 63 64 66 68 69 72 73 75 77 79 81 82 84 86 88 90 ºC 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 From Peters and Van Slyke, Quantitative Clinical Chemistry, vol. 11. (Methods) Baltimore: Williams and Wilkins, 1932, reprinted 1956. www.biopac.com Page 16 of 16