Indirect Calorimetry: From Expired CO2 Production, Inspired O2 Consumption to Energy Equivalent

This paper emphasizes the methodology of data collection of indirect calorimetry, including establishment of steady state conditions and the standards in which the values are presented. It also aims to describe in details the calculations of VO2, VCO2, Resting Energy Expenditure (REE) and Respiratory Quotient (RQ). The trial is registered with ClinicalTrials.gov number NCT02072694.


Introduction
The indirect calorimetry can be understood as a non-invasive measurement of energy produced by the organism through quantifying the volumes of consumed oxygen and produced carbon dioxide (V o2 and V co2 , respectively) by the oxidation of substrates [1].
The calorimeters commercially available calculate V o2 and V co2 through equations using concentrations of O 2 and CO 2 concentrations in the inhaled air (FIO 2 and FICO 2 ) and exhaled air (FEO 2 and FECO 2 ), respectively, and the inhaled (VI) and exhaled (VE) lung volumes of air per minute [2,3].
While the software acquired with the calorimeter provides all results, understanding how the calculations from which the values of V o2 , V co2 and Resting Energy Expenditure (REE) are obtained could be challenging even for experts in this field. Therefore, this report shows how the data provided by the calorimeter could be used to compute those values, since the establishment of steady state conditions, once seldom papers clearly demonstrate it.

Subject
A 34 years-old healthy female volunteer, BMI 18.7 kg/m 2 , underwent an indirect calorimetry test. The subject was a participant of a study approved by the ethical review board of Ribeirão Preto Medical School of São Paulo University, which is registered with ClinicalTrials.gov number NCT02072694.

Experimental design
According to the manufacturer recommendations, the calorimeter Quark RMR® (Cosmed, Rome, Italy) was turned on 45 minutes before calibration [4] in 3 steps: 1) Ambient air; 2) standard mixture composed of CO 2 , O 2 and N 2 in the concentrations of 5%, 16% and 79%, respectively and 3) Validation of the bidirectional digital turbine flowmeter, performed using a certified 3 L calibration syringe [5]. The IC test was performed as suggested by Compher et al. [6] and Suen et al. [7], with canopy, being the flow rate regularly adjusted to maintain a constant FECO 2 through all the time.
Before the acquisition of data, the participant was asked to remain quiet, awake, with a regular respiratory pattern, avoiding yawning, coughing, speaking and sighing. After emptying of the bladder, her height and weight were measured; the volunteer layed down on supine position with her head elevated 30 degrees, extended limbs and opened eyes in a silent room at 23°C [7].
The indirect calorimetry test lasted approximately 15 minutes, in which was assured that all recommendations were followed.

Establishment of steady state conditions and data analysis
Initially, the obtained data was exported from Cosmed 10.0a software to a spreadsheet of Microsoft Excel 2007® and the first 5 minutes were not taken into account. In the graphic plotted with the variations of V o2 and V co2 along the remaining 10 minutes, a 5-minute period with steady state conditions was chosen. In this interval, the coefficients of variation of the observed V o2 and V co2 were respectively 4.8 and 4.7 [5] (Figure 1). The mean values of VE, FEO 2 and FECO 2 were calculated and used in the equations to obtain the V o2 , V co2 , REE and RQ. The volume of VE was provided by the calorimeter in L/min BTPS, which means the volume of a gas at body temperature (37°C), ambient pressure and saturated with water vapor at the subject's body temperature [8].

Discussion
The indirect calorimetry has been used as a research and clinical practice tool [10]. Usually, the software provided with the available calorimeters processes the collected data and summarizes it in reports or spreadsheets containing the values of REE, V o2 , V co2 , RQ and others.
Nevertheless, sometimes the researcher wishes to understand how this software computes the aforementioned values. Once, in the literature, there are discrepancies among the equations used [9,11], some difficulties arise at this point. Moreover, researchers must attempt to not only describe the values given by the calorimeter software, but also to report the methods to obtain it in order to assure the quality of collected data. Therefore, it is important to disclose all equations used, how steady state conditions were established; the standard of the values (BTPS or STPD) and under which conditions the test was executed.
The value of VE given by the calorimeter and the VI, in this case, calculated through Haldane's transformation are expressed in BTPS. However, the values of V o2 and V co2 are usually expressed in STPD, which is the volume of a gas under standard conditions of temperature (0°C), barometric pressure (760 mmHg) and humidity (partial pressure of water, 0 mmHg) [8]. Therefore, it is necessary to adjust the V o2 and V co2 to STPD.
Because detailed reports about the calculations related to indirect calorimetry are commonly not found, this paper illustrates all the steps involved, since the arrangements for test execution, the establishment of a steady state interval, the calculations of the values of the V o2 , V co2 , REE and RQ. In addition, it emphasizes the adjustment of values to STPD.