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• #### 9th February 2021

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Lesson 6, Topic 1
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# Direct and indirect calorimetry

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### Methods adopted to estimate energy requirements for maintenance:

Energy requirements are best determined by measurement of energy expenditure. Energy expended for maintenance of an animal is converted into heat and leaves the body. Thus, an intake sufficient to offset the loss represented by the fasting metabolism would be the requirement if the animal is maintained under basal conditions.

### Fasting Metabolism as a Basis for Estimating Maintenance Requirement:

Dry non-producing, mature animals were fasted, kept in a thermoneutral environment and their heat production was determined (fasting catabolism). This gives an estimate about the minimum quantity of net energy which must be supplied to the animal to keep it in energy equilibrium. This can be estimated by both direct and indirect calorimetry.

### Direct Calorimetry

• Direct calorimetry obtains a direct measurement of the amount of heat generated by the body within a structure large enough to permit moderate amounts of activity. These structures are called whole-room calorimeters.

• Direct calorimetry provides a measure of energy expended in the form of heat. The technique of direct calorimetry has several disadvantages. The structure is costly, requires complex engineering, and appropriate facilities are scarce around the world.

• Subjects must remain in a physically confined environment for long periods. In addition, direct calorimetry does not provide any information about the nature of substrates that are being oxidized to generate energy within the body

• This is simple in theory, difficult in practice; sensible heat loss (heat of radiation conduction) from the animal body can be measured with two general types of calorimeters, adiabatic and gradient.

• The insensible heat (latent heat of water vaporized from the skin and the respiratory passages) is estimated by determining in some way the amount of water vapour added to the air, which flows through the calorimeter. For this, rate of airflow and change in humidity is measured.

### Adiabatic Calorimeters

• In this type an animal is confined in a chamber constructed in such a way that heat loss through the walls of the chamber is reduced to near zero. This is attained by a box within a box.

• When the outer box or wall is electrically heated to the same temperature as the inner wall, heat loss from the inner wall to the outer wall is impossible.

• Water circulating in a coil in such a chamber absorbs the heat collected by the inner wall; the volume and change in temperature of the water can be used to calculate sensible heat loss from animal body.

• The construction and operation are complicated and very expensive.

### Gradient Calorimeters

• Calorimeters of this type allow the loss of heat through the walls of the animal chamber.

• The outer surface of the wall of the calorimeter is maintained at a constant temperature with a water jacket; the temperature gradient is measured with thermocouples, which line the inner and outer surfaces of the wall.

• By the use of appropriate techniques, it is possible to measure separately the radiation component of the sensible heat loss.

### Indirect Calorimetry

In indirect calorimetry, energy expenditure is determined by measuring the amount of oxygen consumed and carbon dioxide produced. Indirect calorimetry is the method by which measurements of respiratory gas exchange (oxygen consumption, V O 2 and carbon dioxide production, V CO 2 ) are used to estimate the type and amount of substrate oxidized and the amount of energy produced by biological oxidation.

• The respiratory quotient (RQ), which provides information about metabolic substrate utilization (lipid or carbohydrate), is calculated by dividing the volume of CO2 produced by the volume of O2 consumed (RQ = VCO2/VO2).

• Indirect calorimeter apparatus are ventilated, open-circuit systems. Rats or mice are housed in a gas-tight metabolic cage through which a flow of fresh air is passed.

• The system collects and mixes the expired air, measures the flow rate, and analyzes the gas concentration of the incoming and outgoing air for both O2 and CO2. Alternatively, although less accurate, the doubly labeled water method, an indirect method of calorimetry based on isotope-elimination, can be used.
• Animal body ultimately derives all of its energy from oxidation, the magnitude of energy metabolism can be estimated from the exchange of respiratory gases.

• Such measurements of heat production are more readily accomplished than are measurements of heat dissipation by direct calorimetry.

• A variety of techniques are available for measuring the respiratory exchange; all ultimately seek to measure oxygen consumption and CO2 production per unit of time.

### 1. Open Circuit System:

• Devices allow the animal to breath atmospheric air of determined composition; the exhaust air from a chamber or expired air from a mask or cannula, is either collected or else metered and sampled and then analysed for O2 and CO2 content.

• Analysis of gases has been accomplished with chemical and volumetric or manometric techniques.

### 2. Closed Circuit System:

• Devices require the animal to rebreathe the same air.

• CO2 is removed with a suitable absorbent which may be weighed before and after use to determine its rate of production.

• The use of oxygen by the animal body decreases the volume of the respiratory gas mixture, and this change in volume is used as a measure of the rate of oxygen consumption.

• Oxygen used by the animal is then replaced by a metered supply of the pure gas.

• Both O2 consumption and CO2 production must be corrected for any difference in the amounts present in the circuit air at the beginning and end of the experiment.

• Methane is allowed to accumulate in the circuit air and the amount present is determined at the end of the experiment.

### 3. Indirect Calorimetry by the Measurement of Respiratory Exchange:

1. The substances which are oxidised in the body, and whose energy is therefore converted into heat, fall mainly into the three nutrient classes of carbohydrates, fat and proteins.
2. In an animal obtaining all its energy by the oxidation of glucose, the utilisation of 1 litre of oxygen would lead to production of 673/(6×22.4)=5.007 Kcal of heat, for mixtures of carbohydrates the average value is 5.047 Kcal, and for mixtures of fats alone, the average value is 4.715 Kcal per litre.
3. Such values are known as thermal equivalents of oxygen, and are used in indirect calorimetry to estimate heat production from oxygen consumption.
4. They oxidise a mixture of Carbohydrate & Fat (and of protein also), so that in order to apply the appropriate thermal equivalent when converting oxygen consumption to heat production it is necessary to know how much of the oxygen is used for each nutrient. The proportions are calculated from what is known as the respiratory quotient (RQ). This is the ratio between the volume of carbon dioxide produced by the animal and the volume of oxygen used.
5. RQ can be calculated from the molecules of carbon dioxide produced and oxygen used. RQ for carbohydrate is calculated as 6 CO2/6 O2 = 1, and RQ of the fat, as 51 CO2/72.5 O2=0.70. For each gram of protein oxidised, 0.77 litres of carbon dioxide is produced and 0.96 litres of oxygen used, giving an RQ of 0.8.
6. If the RQ of an animal is known, the proportions of fat and carbohydrate oxidised can then be determined from standard tables. For example, an RQ of 0.9 indicates the oxidation of a mixture of 67.5% carbohydrate and 32.5% fat, and the thermal equivalent of oxygen for such a mixture is 4.924 Kcal/litre.

7. The quantity of protein catabolised can be estimated from the output of nitrogen in the urine, 0.16g of urinary N being excreted for each gram of protein.