Laboratory Methods

Whole Body Indirect Calorimetry

The advent of indirect calorimetry was a significant event in the history of animal and human nutrition. In whole body indirect calorimetry, the subject is kept in a sealed room or chamber, which is ventilated with a constant, measured supply of air. 'It is a setting similar to his habitual living and hence a more applicable measurement of energy expenditure.' The respiratory gas exchange of the subject is measured by the change in composition of the air going into the chamber and that of the air expelled from the chamber. Well-mixed samples of the chamber air are drawn to be analyzed for chamber air composition. The difference in O2 and CO2 composition of the incoming and outgoing chamber air is used to calculate the energy expenditure and macro-nutrient oxidation of the subject. Two main types of indirect calorimetry systems exist.

Closed-circuit indirect calorimetry involves the recirculation of the same air through the chamber. This can be performed by placing the subject in a sealed chamber. The recirculated air is kept breathable by removing the CO2 produced by the subject and replacing the O2 consumed by the subject. The replacement of O2 is controlled by continuously monitoring the change in the volume of the gas in the closed breathing circuit. As the subject consumes O2, a sensor detects the decrease in volume and a signal is sent to an external source to release constant calibrated pulses of O2 back into the system to restore the original values. The rate of O2 consumption is measured by recording the amount of O2 that is added to the air during recirculation. The CO2 produced by the subject is removed from the recirculated air by an absorber attached to the system and the CO2 production is measured from the increased weight of the absorber (Figure 1).

Open-circuit indirect calorimetry involves a system in which both ends of the breathing system are open to the atmosphere. The inspired and expired air are kept separate by means of a three-way respiratory valve or non-rebreathing mask. The expired gases are collected into an air-tight bag or are frequently sampled or continuously analyzed for O2 and CO2 content.

These two terms are also used to describe some of the many other forms of smaller indirect calorimeters that have been developed over the years.

External O2 supply

External O2 supply

Pressure sensor

Figure 1 Closed-circuit metabolic chamber in which the subject's oxygen consumption is measured to calculate the corresponding energy expenditure. The change in volume of air in the system is constantly monitored by the sensors and a measured quantity of oxygen is added back to the system. Carbon dioxide is taken out of the recirculated air by a CO2 absorber.

Pressure sensor

Figure 1 Closed-circuit metabolic chamber in which the subject's oxygen consumption is measured to calculate the corresponding energy expenditure. The change in volume of air in the system is constantly monitored by the sensors and a measured quantity of oxygen is added back to the system. Carbon dioxide is taken out of the recirculated air by a CO2 absorber.

These can be categorized as laboratory or field techniques based on their portability. The instrumentation used for each varies in complexity and the degree to which they restrict the subject's movement.

Metabolic Carts

Metabolic cart is a common name for a semiport-able respiratory gas analyzer that has been made small enough to be placed on a cart with wheels so that it can be rolled to different locations within a building. Two designs are generally available: the ventilated hood and the mouthpiece system.

The ventilatory hood system is an open-circuit indirect calorimeter that usually consists of a pliable plastic or rigid Perspex hood placed over the subject's head with a latex or thin plastic apron providing a rough seal around the neck or chest. These allow air to be drawn across a subject's face while in a reclining or lying position. For longer term measurements, ventilated plastic tents are available that cover all or part of the patient's bed. Since these hoods operate on a suction principle, a tight seal of the hood is not required. For field measurements, whole body transparent plastic ventilated boxes have been used successfully in infants. Many of the ventilatory hoods are constructed by researchers from the components according to the requirements of their study. The components include a pump, a flow meter, and a means of regulating the airflow. Samples of the air drawn from the hood can be directed to gas analyzers, which are usually connected in series to the hood. Respiratory gas exchange is calculated from the difference in O2 and CO2 concentration between the air entering and exiting the hood and the controlled rate of airflow (Figure 2).

Instruments have been developed to operate in adult and pediatric applications and differ with respect to flow rates and internal volume because of the smaller metabolic rate of children. The expired air enters a mixing chamber within the instrument to eliminate concentration variation resulting from inspiration and expiration before the sample enters O2 and CO2 sensor analyzers, which measure the concentration differences between the expired and inspired air. For state-of-the-art instruments, the data are input into a microprocessor providing minute-by-minute calculation of the O2 consumption, CO2 production, RER, and energy

Hood (subject)

Flow meter

Mixing chamber

O2

CO2

analyzer

analyzer

Figure 2 Ventilatory hood system showing a hood that is placed on the subject's head, a mixing chamber, and O2 and CO2 analyzers. A fan maintains a slight negative pressure in the hood to pull air into the chamber and also to prevent the escape of the expired air from the system. The air is mixed in the mixing chamber and is analyzed for oxygen and carbon dioxide by the respective analyzers. Results are calculated by the computer.

Figure 2 Ventilatory hood system showing a hood that is placed on the subject's head, a mixing chamber, and O2 and CO2 analyzers. A fan maintains a slight negative pressure in the hood to pull air into the chamber and also to prevent the escape of the expired air from the system. The air is mixed in the mixing chamber and is analyzed for oxygen and carbon dioxide by the respective analyzers. Results are calculated by the computer.

Mixing chamber

Diluting chamber

Expired air-inlet Expired air-inlet

From mouth piece

Ambient air-inlet

To O2

To CO2

analyzer •

Figure 3 Metabolic unit measuring both O2 consumption and CO2 production rates during rest and exercise. In this type of system, expired air is diluted using ambient air before being analyzed by the respective analyzers.

expenditure. These instruments are generally used for measurements of subjects at rest as part of nutritional studies of energy expenditure and macronu-trient utilization. These units can also be connected to mechanical ventilators for use in hospitalized patients.

Mouthpiece systems are similar to ventilated hood systems in principle, but instead of placing a hood over the subject's head, the subject wears a mouthpiece connected to the analyzer and nose clips to prevent breathing through the nose. The mouthpiece is connected to a valve system that allows the subject to breath in atmospheric air while directing the exhaled air to the gas analysis system. The expired breath is again subjected to analysis of O2 and CO2 concentration, but rather than passing the breath through a mixing chamber to smooth out the changes in concentration gradient of these gases from the start to end of an exhalation, the concentration profile is measured in real time along with the rate of gas flow from the exhalation. Again, the data are fed into a microprocessor for calculation of O2 consumption and CO2 production, but in this case the calculation is performed on a breath-by-breath basis. Results are averaged over time, usually provided as minute-by-minute averages of O2 consumption, CO2 output, and the rate of energy expenditure. The mouthpiece systems are generally used for studies of gas exchange and energy metabolism during exercise and provide a shorter measurement response time than the ventilated hood systems. The mouthpiece and nose clip used with some of the instruments make long time measurements highly cumbersome. Also, breathing through the mouthpiece often causes untrained subjects to involuntarily hyperventilate leading to inappropriate O2 and CO2 rates. It is also often difficult with mask systems to obtain an airtight seal without excessive pressure at the site of contact with the mask and face.

Different types of metabolic carts or monitors are available that are designed for various applications ranging from nutrition to exercise science. Most have built-in gas analyzers and data processing computers, making them highly user-friendly, handy tools for measurement of energy metabolism. They generally provide accurate and reliable data but do require periodic calibration. Ventilated hood systems often use a combination of gases with known concentrations and weighed ethanol or methanol burns for such calibration, whereas breath-by-breath systems use a combination of large volumetric syringes and gases of known O2 and CO2 concentration (Figure 3).

Field Methods

As for whole body indirect calorimetry, ambulatory and portable systems measure the respiratory gas exchange with the VOl and VCO2 measurements. Ambulatory methods and less refined laboratory methods often dispense with the measurement of CO2 to avoid the need for two gas analyzers. The error incurred by assuming a CO2 production rate is several percentage points, which researchers are prepared to compromise on. When only O2 consumption is measured, however, it is not possible to compute macronutrient-specific oxidation rates. The accuracy of ambulatory and portable methods is generally between +4 and —2%. Field methods involve the collection of expired air over a fixed period of time as in the Douglas bag or small online analysis systems that sample inspired and expired air through a mouthpiece.

Douglas Bag/Tissot Tank

The Douglas bag method is a classical example of collection of expired air to measure energy expenditure in the field during both rest and physical activity. It consists of a gas-impermeable bag with a capacity of ~100l or a Tissot tank suspended over water, which is used to store the subject's expired air over a fixed, short time interval. A classic Douglas bag is made up of either a rubber sheeting cemented by two layers of canvas or plastic material lined by PVC or aluminum with welded seams. The rubber bags have leakage of CO2 by diffusion, which is unavoidable, but PVC and metalized bags hold better. If the bags are filled to capacity and analyzed with 20 min of collection, the effects of diffusion can be minimized. The subject wears a nose clip and mouthpiece or a face mask. Outside air or its equivalent is inhaled through the mouthpiece or mask containing a oneway valve and exhaled into a Douglas bag or Tissot tank for a precise period of time. It is important that the mouthpiece and connecting tubing provide minimal resistance to airflow, or the cost of breathing will increase the energy expenditure. Ambient temperature, barometric pressure, and relative humidity are recorded for converting values under conditions of standard temperature and pressure. The volume of air collected in the bag or tank is measured and a sample of exhaled air is obtained to measure the O2 and CO2 concentrations using gas analyzers. The volume of oxygen consumed and carbon dioxide exhaled are calculated by analyzing the gas from the Douglas bag for the precise time period during which it was collected. This method is relatively simple and inexpensive yet gives reliable results. It is suitable only for short durations of field measurement, and wearing the mask and nose clip for the whole duration of the study may be cumbersome, interfere with daily activities, and is socially undesirable to the subject.

Spirometers were used in the past for measurement of the volume of the respired air. With the advent of continuous flow electronic analyzers and superior gas flowmeters, spirometers are now rarely used. Ambulatory methods also consist of a mouthpiece incorporating light action-sensitive but robust one-way gas valves, corrugated tubes, and three-way taps. The volume of air respired and the relative concentrations of O2 and CO2 in the expired air are measured using O2 and CO2 gas analyzers. These small analyzers have replaced the Haldane system or micro-Scholander chemical gas analyzers, which used reagents to absorb the CO2 and O2, with the weight of absorbents measured before and after the gases were absorbed.

Max Plank/Kofranyi-Michaels Respirometer

A Max Plank respiration gas meter is a small, compact, and lightweight backpack-mounted respi-rometer. It combines a gas volume meter and a sampling device for continuous sampling of each breath of expired air. The Max Plank respirometer consists of a dry, bellow-type gas meter for measuring the total volume of expired air during activity. The subject breathes through a low-resistance valve and the expired volume is monitored. A measured quantity of expired air is removed continuously (0.3 or 0.6%) by an aliquoting device to be sent to a small butyl rubber bag. This rubber sampling bag can be connected directly to the oxygen analyzer, eliminating the need for transfer of samples to gas-tight syringes for analysis. The respirometer is suitable for flow rates between 15 and 50l/min or for periods of 110 min on a slow flow rate and 55min on a faster rate. It is smaller, more compact, and lighter than the Douglas bag apparatus and can be used in studies involving light to moderate physical activity. Although the system has a low resistance, at higher ventilation rates the resistance increases substantially and hence cannot be used in higher flow rate scenarios. Also, this can be used in studies of shorter duration only. Due to the use of mouthpiece and nose clip, prolonged usage may cause discomfort to the subjects.

Telemetry Systems

The K2 system was the first of a series of portable systems that consists of a soft face mask with a turbine flowmeter attached to it. A transmitter and battery are attached to a chest harness, which transmits signals to a receiver unit. The flowmeter measures the rate of airflow, calculates the volume of expired air per minute, and counts the number of expirations per minute. A small capillary tube passes through to the transmitter unit, which contains an electrochemical gas analyzer used to measure the concentration of oxygen in expired air. The signals from this analyzer are transmitted to the receiver unit by the portable transmitter unit. The receiver unit processes the data and prints it in a desired format. The electrochemical gas analyzer is a polarographic electrode. It has a membrane through which oxygen permeates into an electrolyte solution generating an electrical impulse proportional to the rate of oxygen permeation through the membrane.

Since these systems are portable and easy to use, they have many potential uses in exercise science studies and rehabilitation medicine. They allow a breath-by-breath pulmonary gas exchange

Figure 4 Telemetry system with a face mask attached to a turbine flowmeter, a transmitter, and a receiver unit. The flowmeter measures the rate and volume of airflow and the expiratory cycles per minute. The expired air is analyzed for oxygen concentration by an oxygen analyzer in the transmitter unit. The transmitter then transmits the signals to the receiver unit, which integrates the data and prints the results.

Figure 4 Telemetry system with a face mask attached to a turbine flowmeter, a transmitter, and a receiver unit. The flowmeter measures the rate and volume of airflow and the expiratory cycles per minute. The expired air is analyzed for oxygen concentration by an oxygen analyzer in the transmitter unit. The transmitter then transmits the signals to the receiver unit, which integrates the data and prints the results.

measurement while still being very light and portable, enabling a direct field assessment of human performance and cardiopulmonary limitations. The low-resistance flowmeter allows a wide range of oxygen flow rates to be measured, through these systems face the issue of air leakage from the face masks when subjects are made to exercise at high intensities. The measurement durations usually are limited to 1-5 h. The polarographic electrode membrane is known to have a short life span and hence monitoring of the usage of the instrument is essential. If CO2 concentrations are essential for a study, this is not a good instrument to use (Figure 4).

Tracer Methods of Indirect Calorimetry

These are a third category of techniques that have gained popularity among investigators during the past two decades. These techniques provide a measure of CO2 production through the use of dilution techniques using isotopic tracers.

Labeled Bicarbonate

A constant infusion-labeled bicarbonate method is useful in estimating the net CO2 production and hence energy expenditure in animals and humans. This method is based on an isotopic dilution technique whereby the administered label is diluted by the CO2 produced endogenously by the body. The extent of this isotope dilution is used to measure the rate of CO2 production and is used to estimate the energy expenditure of the individual. A microinfusion of 13C- or 14C-labeled bicarbonate is given to an individual and the specific activity or enrichment of his or her physiological fluids, especially breath or urine, are measured to estimate the rate of label elimination and hence the rate of endogenous CO2. Thus, variation in the endogenous CO2 production rate will be reflected in the dilution of the body pool and consequently in the breath samples.

These measurements are accurate when energy expenditures are measured over a longer duration of time (>1day) but are subject to effects of label sequestration over shorter periods. Sequestration refers to trapping, or fixation, of the label in tissues that utilize bicarbonate/CO2 for their metabolic functions. Shorter duration of collection of breath samples requires a correction for the fraction of label that is sequestered. This is based on the assumption that similar amounts of label are sequestered in various individuals. When breath samples are collected over longer durations, the sequestration is often assumed to be negligible.

Some investigators have used a bolus bicarbonate administration rather than the continuous infusion. These investigators measured the rate at which the label concentration decreases with time as a measure of CO2 turnover and the initial concentration as a measure of the body's bicarbonate pool size. Taken together, these provided a measure of energy expenditure during a short period of constant physical activity.

Doubly Labeled Water

This is an isotope dilution technique wherein deuterium and heavy oxygen-labeled water (doubly labeled water, DLW) are given to individuals and timed urine samples are collected to measure the elimination rates of 2H and 18O in the urine. 2H label from DLW mixes with the body water and is eliminated as water in the urine. Similarly, 18O label from DLW is eliminated as water, but it is also utilized in bicarbonate synthesis and hence is also eliminated in the breath as CO2. The difference in turnover rates of isotopic 2H-H and 18O-labeled water is proportional to CO2 production. Energy expenditure, oxygen consumption, water intake, and metabolic water production can be calculated using standard indirect calorimetry equations with an estimated RER (Figure 5).

In practice, a measured dose of DLW is given to the subject whose energy expenditure is to be measured. Body water samples, such as blood, urine, saliva, or breath water, are collected before dosing and after equilibrium is attained. The isotopic disappearance rates of 18O and 2H as CO2 in breath or

Figure 5 Time course on log scale for the enrichments of the stable isotopes 18-oxygen and deuterium when administered to the subject. Both the tracer enrichments increase rapidly in the body water pool until they reach distribution equilibrium (2-4 h). The enrichments then start to decline as the body water turns over during metabolism. 18-Oxygen is eliminated at a faster rate because it is excreted as water and CO2 in breath, whereas deuterium is eliminated as water only. The difference in elimination rates of these two tracers is proportional to the rate of CO2 production by the subject.

Figure 5 Time course on log scale for the enrichments of the stable isotopes 18-oxygen and deuterium when administered to the subject. Both the tracer enrichments increase rapidly in the body water pool until they reach distribution equilibrium (2-4 h). The enrichments then start to decline as the body water turns over during metabolism. 18-Oxygen is eliminated at a faster rate because it is excreted as water and CO2 in breath, whereas deuterium is eliminated as water only. The difference in elimination rates of these two tracers is proportional to the rate of CO2 production by the subject.

H2O in urine, saliva, or breath water, respectively, are determined from the change in isotopic enrichments of the before dosing and after equilibrium samples.

The doubly labeled water method is both simple and noninvasive. It has been validated in various animals and humans, with the CO2 production rate showing a mean measurement error of less than 5%. Unlike the majority of the other methods, the doubly labeled water method provides a measure of average energy expended over a period of 3-21 days without restricting the subject's movement and thus provides a better estimate of habitual energy expenditure than the other methods. The doubly labeled water method, however, does not provide any information on the pattern or intensity of any one activity during that time but the overall average energy expenditure. This method is also expensive due to the cost of the 18O and it does require sophisticated mass spectro-metric analyses.

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