METABOLIC RATE LABORATORY
I . Introduction
The problem: You need to determine the amount of food that must be taken per day by
two astronauts for a mission that lasts for two weeks. The astronauts need to be kept happy,
since you have been selected to be one of the astronauts. You and your partner do not wish to
gain or lose weight. When you report to Mission Control at NASA, you will need to tell them
several things, which should be in your report. Much of this will come from your laboratory
measurements, but you may need other resources also. The details about the content of the
report are at the end of this lab manual.
The total process of cellular respiration in any organism using glucose under aerobic
conditions can be summarized by the following equation:
C6H1206 + 6 02 => 6 C02 + 6 H20
G = - 686 Kcal/mol of glucose
36 ADP + 36 Pi => 36 ATP
G = +262.8 Kcal/mol of glucose (7.3 Kcal/mole of ATP)
These are the values of G under standard conditions. Remember that one calorie is the
amount of energy needed to heat one gram of water by one degree C. A mole of glucose (180
grams) could, therefore theoretically heat 6.86 Kg of water from the freezing point to the boiling
point!
The rate at which an organism utilizes energy, measured in Kcal/ hr or other units of
energy per time is metabolic rate, and if measured in Kcal/kg - hour or similar units of
energy/mass-time can be considered specific metabolic rate. The rate at rest is the basal
metabolic rate (BMR), defined for humans to be the metabolic rate that is found after a person
has fasted for 12 hours, slept for at least 8 hours, and has been resting quietly for at least half
an hour. Muscular activity will, of course, increase the metabolic rate. Since the conditions for
BMR are difficult to achieve, we will really be measuring Resting Metabolic Rate (RMR), which
does not require the fasting and half hour resting, and is expected to be a little higher.
Metabolic rate can be measured by measuring the temperature change of a closed
container containing a metabolizing organism, since the temperature change and heat
production are just related by a proportionality constant – the heat capacity. This is called direct
calorimetry. It is accurate, but requires very specialized equipment so it is inconvenient,
especially for large animals. Since living organisms use molecular oxygen only for cellular
respiration, the rate of oxygen consumption is directly related to the rate of ATP production.
Also, since oxygen is not given off as a by-product of other cellular reactions (except, of course,
photosynthesis), it must be obtained from the environment. Because oxygen consumption from
the atmosphere can be easily and accurately measured without resorting to complicated
technical analytical procedures, metabolic rates of terrestrial organisms can be obtained. In this
lab we will take advantage of the stoichiometric relationships between energy production and
oxygen consumption, and measure oxygen consumption. This is called indirect calorimetry,
which has several variants.
In order to relate oxygen utilization to metabolic rate, we need to estimate what
substrates are actually being used. We really need a value for K, the number of Kcal liberated
for conversion to ATP by 1 Liter of oxygen.
For glucose we know that if we use up 6 O2 molecules or produce 6 CO2 molecules we
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make about 36 ATP molecules. So, for glucose, we have (686 Kcal/mole) / (6 moles of
O2/mole of ATP) = 114.33 Kcal/mole of oxygen. At STP (zero centigrade and 1 atm), 1 mole of
oxygen = 22.4 liters (this is derivable from PV=nRT), so
Kglucose = 114.33/22.4 = 5.104 Kcal / L of oxygen
Again, this is true only if we are metabolizing only carbohydrate. If we metabolize fat, for
instance one of the more common fatty acids, palmitic acid, the reactions are
CH3(CH2)14COOH + 23 O2  16 CO2 + 16 H2O
129 ADP + 129 Pi  129 ATP
-2340 Kcal/mol
+942 Kcal/mol
We can easily derive that K will be:
Kfat = 4.7 Kcal / L of oxygen
If we metabolize protein, which usually makes up a small component of the diet, we can't
write a single reaction. Some amino acids are converted to alanine before energy is derived,
and alanine is usually the amino acid in the highest concentration in the blood:
2 C3H7O2N + 6 O2  (NH2)2CO + 5 CO2 + 5 H2O
In addition to CO2 and water, amino acid catabolism produces urea, whose production is
expensive energetically. Protein varies in its caloric value but we will take the average to be
Kprotein = 4.6 Kcal / L of oxygen
What do we do if we have a mixed diet containing all substrates? To consider this, we
define the respiratory quotient:
Respiratory quotient = R.Q. = Moles of CO2 produced / Moles of O2 consumed
From the stoichiometry again, R.Q for carbohydrate is 1.0, for fat it is 0.703 and for protein it is
0.833. It turns out that:
K (Kcal/L of oxygen) = (3.94 + 1.11 RQ)/(1 + 0.082 P)
where P is the fraction of calories derived from protein. This is called the Weir equation. If you
measure RQ and know P you can get a value for K (Kcal/L) from the Weir equation exactly.
As an aside, RQnet = C * RQC + F * RQF + P * RQP
C, F, P are the fractions of calories derived from each substrate (not the fraction of mass of
each substrate), so the actual RQ is a linear combination of the RQs of each substrate. You
may wish to calculate how much K varies if the calories derived from protein vary. It turns out
that a measurement of RQ alone gives a relatively good value for K. Note that the value of RQ
in the Weir equation is the actual value of RQ. This need not be the same as the RQ calculated
from the calories in the diet, because RQ will be determined not by what you just ate, but what
substrates are being used for metabolism. These could be the same, but are not guaranteed to
be the same. For instance, if you eat only carbohydrates, the RQ calculated from the diet would
be 1, but some tissues will still metabolize some fatty acid, so the actual RQ would be a little
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less than 1.
We will not attempt to get an exact value for K, and will instead assume the value often
used for a fasting person. As you might expect, this is between the values for carbohydrate and
fat.
Our K = 4.82 KCal / L O2
As another aside, efficiency should really be measured by considering the energy
recovered as ATP divided by the energy available from the substrates. In this formulation, the
efficiency of both carbohydrate and fat oxidation is similar, about 40%. We often discuss
efficiency as energy available per gram of substrate. In those units:
Carbohydrate:
4 Kcal/gram
Fat:
9 Kcal/gram
Amino acids:
~ 4 Kcal/gram (range is 2.12 Kcal/gram (glycine) to 5.9 Kcal/gram
(leucine))
Fat is presumably the main way we and migrating birds and hibernating bears store energy
because we get more energy per unit weight. The difference is even bigger than the numbers
might appear, because fat is stored as pure fat, where as carbohydrate and protein have to be
dissolved in about three times their weight in water.
II. Human measurements
We will not quite meet the conditions needed for measuring BMR, so we are measuring RMR,
resting metabolic rate, instead.
In this lab we will be measuring human metabolic rate by indirect calorimetry – that is, with
oxygen consumption. We recognize that
Vol O2 consumed = Vol O2 inspired – Vol O2 exhaled
= FIO2 * VI – FeO2 * VE
Where FeO2 and FIO2 are the fractions of oxygen in expired and inspired gas. After obtaining
Vol O2 consumed, we use the conversion factor above, K, to determine Kcal/hr.
We obtain FIO2 from known values in the atmosphere: 20.93% in dry air. We obtain FeO2 from
an oxygen sensor (oxygen electrode). An oxygen electrode is an electrochemical device in
which the reaction at the cathode is:
2H2O + O2 + 4 e-  4 OHThis reaction occurs when the cathode is polarized at a negative voltage relative to the anode,
which in the system we will use is lead, but may be other metals. One essentially counts the
electrons produced in this reaction (i.e. measures current) and this is proportional to the oxygen
that reacts. One has to set up the electrode so that all of the oxygen that reaches the cathode
reacts. This can be done by polarizing at about 0.7 V. One always has to create a calibration
curve, but since the current is linearly related to oxygen concentration, a two point calibration
curve is sufficient. Oxygen electrodes using this principle, but with slight modifications, are used
to measure oxygen in many settings. Blood-gas machines in hospitals use this technique to
measure oxygen in arterial blood.
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We obtain VE by collecting expired gas in a "Douglas bag"; we then run this through either a
spirometer, a homemade displacement device, or a dry gas meter. We have a one-way valve
so that inspired and expired gas do not mix.
We will assume that VI = VE. There is less than 2% error in this, even if we are using fat
metabolism and RQ = 0.7. (There is no error if RQ = 1.) For research in this area a slightly
different technique is used to reduce the error even further.
It will be interesting to compare our results to those in the literature. Accepted relations for
basal metabolic rate are:
Males: Kcal/24 Hr = 66.473 +13.75 W + 5.003 H – 6.755 A
Females: Kcal/24 Hr = 655 + 9.563 W + 1.85 H – 4.676 A
Here W is weight in Kg, H is height in cm (not meters), and A is age in years. The male and
female equations look very different, but they are both correct.
(Consolazio, CF, Johnson, RE, Pecora, LJ (1963) Physiol. Meas. Of Metabolic Function in
Man. McGraw Hill, NY.)
III. Lab procedures:
The general strategy will be to collect expired gas in Douglas bags, and then to perform
measurements on that gas. You will need to measure:
1 – The time of gas collection under known physiological conditions, either resting or during
stationary bicycle exercise.
2 – Oxygen content of the gas
3 – Total volume of the expired gas.
In addition we should, ideally, measure the relative humidity of room air so that we can obtain
the actual fraction of inspired oxygen, which will be a little lower than 20.93% due to the
presence of water vapor.
Each lab group should make measurements under resting and exercising conditions. It is
preferable to make a “good” set of measurements on one person than making “not so good”
measurements on two people. Group members should not participate in the exercise part
of the lab if they have any health problems.
When you exercise, you can pedal at different rates producing different workloads. The bicycle
measures work, not total energy used. Work is measured in rather odd units on this bike:
kilopond-meters per minute – KPM/M on the dial. A kilopond is a kilogram of force. The bike
actually says "KPM/M *300" This means that the number you read on the dial has to be
multiplied by 300 to arrive at KPM/M. The conversion factor to more familiar units is 0.141
Kcal/hr/KPM/M (or 1 KPM/M = 0.141 Kcal/hr). In order to read KPM/M you must pedal at an
appropriate rate to hold the speed at 60, 75 or 90 revolutions of the pedals per minute. The
dials are color coded and this will become clear in lab. Since you will be measuring efficiency,
you need to record the work level.
For a gas collection episode:
Empty the Douglas bag as far as possible. Attach a headgear apparatus and tubing to the
Douglas bag. Each person will have his/her own mouthpiece containing a filter. Adjust the
headgear and the Douglas bag “backpack.” First set the circular valve plate so that the expired
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gas just goes to room air until you are resting and comfortable with the device. Start timing at
the point at which you rotate the valve plate to begin collection. Close the valve when you stop
timing. Take off the apparatus. Remove the corrugated tubing from the Douglas bag and insert
a stopper instead, being careful not to lose gas.
Oxygen content:
Oxygen content will be measured by oxygen sensors (oxygen electrodes) made by Vernier Co.
They should be kept upright, with the cord facing up. You will insert the sensor into a chamber
made from a modified syringe body that is held in a ring stand, and gas will be allowed to flow
through the chamber. It is necessary to calibrate the sensor before use. Even if another group
has done this, you should check the calibration and adjust it if necessary. The signal will go into
a data acquisition system and computer. There is a computer program all set up for your
measurements. Details will be given in lab. In order to calibrate, you need to blow different
gases by the sensor. For air you can use a 140 ml syringe filled with room air; for zero oxygen,
you can take gas from a tank of pure CO2. Make sure that you get training in the use of the
regulator on the CO2 tank before using it. Gas cylinders are potentially dangerous, since
the pressure in them can be as high as 2200 PSI . Gas from the CO2 tank will push back he
plunger on the calibration syringe. When it is full, disconnect the syringe from the tank, and
close the stopcock on the syringe.
For either gas, slowly inject the gas from the large syringe into the chamber underneath
the gas sensor, and read the voltage produced by the sensor. You may need to check values
more than once. These voltage values give you your calibration. The input window in the
scope program can be used to create the calibration.
Measurement of the expired gas composition is done by using a large syringe to
withdraw air from the Douglas bag. Keep in mind that this reduces the total volume in the bag.
Gas volume measurement. The accuracy of this measurement depends on how well you
emptied the bag before the gas collection, and how well you empty your sample into the gas
meter. Don't forget that you took some gas out for oxygen measurement, and that you are likely
to lose a little during reconnection procedures.
We are using electronic flowmeters made by TSI to measure gas flow from the Douglas bags.
The computer will integrate this flow signal to yield a volume signal. The program is easy to use
and will be demonstrated in lab. Gas needs to be dry before it goes to the flowmeters, so there
is a chamber containing Drierite – a dessicant – preceding the flowmeter.
IV. Some issues to think about before and while doing the lab:
How many measurements do you need to take under resting and exercising conditions in order
to get reliable values?
Should you check over your data before leaving the lab in order to determine whether the data
are good or wait until you are back in your room?
The Douglas bags can hold about 50 L. For how long should you collect the gas under resting
and exercising conditions?
During exercise, will it matter when during exercise you take the sample of expired gas?
During exercise, do you expect the gas content and the expired volume of expired air to change
or just one?
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Do you expect your efficiency (work output/total calories used) to be the same at all work loads?
Do we have to worry about glycolytic metabolism escaping our detection?
V. Lab Report
The format of the lab report is a brief report to NASA, not the standard format (intro,
methods, results, discussion). You are just expected to answer the questions below.
Please use full sentences with good grammar.
A) Mission Control needs a list of the factors that might be expected to influence the caloric
requirements for an astronaut on a mission. Provide a brief explanation of how each factor is
expected to influence caloric requirements.
B) Your measurements have been made on earth. You will not have any other data available,
so you will have to use earthbound data, but in space, astronauts are weightless and there may
be other differences. You will need to explain to Mission Control what you think might allow you
to take RMR data from earth and assume it is correct for conditions in space.
C) Using your own data, what are the total energy requirements for the two astronauts in
Kcal/day, taking into account both resting and exercise periods? Remember that you don't wish
to lose or gain weight on this trip. State any further assumptions you had to make in arriving at
the answer. Present a table of the raw data on which you are basing your calculations. Show a
sample calculation. Compare the RMR portion of the results to the values expected from the
literature, derived from the equations in the lab writeup. You should assume that astronauts will
exercise for two hours per day in space against resistance – similar to the exercise bike. (This
is necessary to minimize bone and muscle loss, but unfortunately some loss cannot be
prevented at present.) You will need to be clear about the level of exercise you are assuming
for the astronauts.
D) As an example, plan one day's meals to meet this energy need, using data on caloric values
in food. This is available in many places: http://www.ntwrks.com/~mikev/chart1.html for one. If
you want to know what astronauts really have to choose from, go to
http://www.jsc.nasa.gov/pao/factsheets/nasapubs/food.html
E) Since you are exercising, NASA is considering the possibility of capturing the work the
astronauts do and using it to charge batteries. This might make the astronauts feel a little like
hamsters, but humor Mission Control, and tell them what efficiency they can expect from a
human powered bicycle.
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