"Metabolism," she writes, "is essentially an oxidation process. The animal uses oxygen in direct proportion to the rate at which it burns foodstuffs for releasing energy to run its internal machinery-energy for growth, for keeping the organism warm, for movement and for running the myriad chemical reactions that constitute the life process. For this reason the amount of oxygen consumed by an organism per unit of time can be used as a measure of its metabolic rate.

"Oxygen consumed by an organism can be monitored in a number of ways. Most hospitals, for example, determine metabolism with a spirometer, an instrument that responds to the volume of oxygen consumed by the patient. Exhaled carbon dioxide, the principal waste product of the metabolic process, is removed chemically. The apparatus includes a pen recorder that automatically draws a graph of the rate at which oxygen is supplied to the patient through a face mask.

"A number of simpler devices have been made for determining the metabolism of small anima]s. Most of the instruments embody the same basic principles. Oxygen is measured in terms of volume consumed per unit of time. Carbon dioxide is removed, sometimes being measured. Certain variables must be taken into account. For example, the animal must be fed normally so that the results of the experiment are not influenced by a diet that is deficient or excessive.

"The rate at which oxygen is consumed reflects the physiological state of the whole animal, the sum total of its chemical reactions. For this reason the experiment can disclose the influence of drugs on a selected chemical reaction within the metabolic scheme. The more significant variables are temperature and barometric pressure. The entire experiment must be run under conditions of controlled temperature and pressure, or these variables must be taken into account mathematically when the data are reduced. The environmental conditions may change substantially during an experiment. For example, marked changes in both temperature and barometric pressure may occur with the approach of a storm. Variables of this kind can be measured by including a thermobarometer in the experiment. It is an apparatus exactly like the one that measures metabolism, but it contains no organisms. Any changes of volume that occur in the thermobarometer system are entered as corrections in the data from parallel runs in which organisms are used.

"One of the most accurate and widely used devices for monitoring oxygen consumption was devised by the German physiologist Otto Warburg. As I have modified it for amateur construction it consists of a sealed animal chamber fitted with a slender glass tube that is open at the outer end [see Figure 2]. As oxygen is consumed by the animal more air flows into the chamber through the manometer tube, carrying with it a droplet of colored water or a strong film of bubble solution to serve as a visual indicator of the gas flow. Car bon dioxide exhaled by the animal is absorbed by a solution of potassium hydroxide on a small wad of cotton. I attach the cotton to the inner end of the tube with a rubber band and then moisten the wad with solution by means of a medicine dropper.

"The potassium hydroxide, which represents 5 percent (by weight) of the solution, is a form of lye that is both toxic and corrosive. Handle it accordingly. If the solution comes in contact with the skin, wash the affected part immediately with lots of water and rinse with vinegar. The chemical will also burn animals on contact. If the animals can crawl up the sides of the bottle or can fly, I enclose the chemical in a protective cage of wire screening.

"I have used this simple apparatus to measure the oxygen consumption of a large variety of small animals, including houseflies, honeybees, fruit flies, earthworms, grasshoppers, crayfish, clams and even fish. Almost any animal can be studied if it is small enough to fit into a wide-mouthed bottle that has a capacity of one or two ounces. Usually I do several experiments simultaneously, using a separate chamber for each animal or group of animals.

"Each bottle is closed with a rubber stopper that is perforated to make a snug fit with a glass tube one to three millimeters in inside diameter. The manometer tube should be about 50 centimeters long, bent to a right angle two centimeters from the stopper so that the long segment extends horizontally when the bottle stands upright. The side of the stopper is coated lightly with Vaseline. After one or more animals have been put in each bottle the stoppers are inserted and he]d in place with a wrapping of waterproof adhesive tape. The bottles are placed in a wire rack that is immersed in a water bath so that the necks of the bottles are just at the waterline. The water has been previously heated or cooled to the desired temperature. If necessary, support the outer ends of the tubes so that they are approximately horizontal.

"After 15 minutes, when the temperature of the air in the bottles has reached the temperature of the surrounding water, I start the experiment by placing a drop of indicator solution (water tinted with food coloring) in the open end of each tube. (A more sensitive indicator that is easier to measure can be made by substituting for the colored drop a film of durable bubble solution [see "The Amateur Scientist," May]. The film is less massive than the drop and hence responds to smaller differences in gas pressure.)

"As the animal continues to respire, oxygen is drawn into the bottle. The volume represented by each one-centimeter length of the tube, multiplied by the distance traveled by the indicator drop as the experiment proceeds, gives the volume of oxygen used. To find the volume of the tube per centimeter of its length, measure the inner diameter of the tube as accurately as possible in centimeters, divide by two and multiply the quotient by itself and by 3.1416. Expressed algebraically, the formula for the volume is V = r²h, in which V is the volume per centimeter of tube length, is 3.1416, r is the radius of the tube and h is its height (in this case one centimeter).

"Include in the rack of immersed animal chambers one empty chamber of identical construction to serve as the thermobarometer. Place the indicator drop inside the thermobarometer tube a few centimeters from the outer end of the tube so that it can move in either direction. Mark the position of the indicator on the glass with a grease pencil. With the apparatus and animals thus prepared, record on a sheet of paper the time when the indicators are placed in the manometer tubes; on the same line enter a zero at the head of a separate column for each bottle in the rack, including the thermobarometer. At 10minute intervals for one hour record for each tube the distance in centimeters that the indicator has moved from its initial position.

"Measure and record the temperature of the water bath in degrees Kelvin. To get degrees Kelvin add 273 to degrees Celsius. If your thermometer is calibrated in degrees Fahrenheit, subtract 32 from the reading, divide the remainder by 9, multiply the quotient by 5 and add 273 to get degrees Kelvin.

"Measure and record the barometric pressure in torr. If your barometer is calibrated in millimeters of mercury, simply record the reading. (One torr is equal to the pressure exerted by a column of mercury one millimeter in height.) If the instrument is calibrated in inches of mercury, multiply the reading by 25.4 to get the pressure in torr.

"From these data you can find by a few simple calculations the amount of oxygen each animal consumes. The actual consumption may vary with the pressure of the atmosphere and with the temperature. It is useful for comparing experimental results observed at various times and under various conditions to adjust the data to a standard barometric pressure and temperature. By agreement biologists use as standards a barometric pressure of 760 torr and a temperature of 273 degrees K.

"First find the apparent volume of oxygen consumed by the animal or animals in each bottle during each of the six 10 minute intervals To find the apparent oxygen consumption in cubic centimeters multiply the distance in centimeters that the indicator moved during each 10-minute interval by the previously determined volume per centimeter of the length of the tube. Determine for each interval of time the change in volume of the air that took place in the thermobarometer. The volume of air in the thermobarometer may have increased (indicated by the outward movement of the colored drop or the bubble film), decreased (indicated by the movement of the indicator toward the bottle) or both.

"Correct the apparent oxygen consumption to the actual consumption by adding or subtracting from each figure the change that occurred simultaneously in the volume of air in the thermobarometer. Record the actual consumption of oxygen for each bottle during each of the six time intervals. Finally, adjust the actual consumption to standard pressure and temperature.

"For each bottle and time interval multiply the actual consumption of oxygen in cubic centimeters by .36 and by the barometric pressure in torr and divide the product by the recorded temperature in degrees Kelvin. Expressed algebraically, the conversion formula is V' = .36VP/T, in which V' is the volume of oxygen consumed at standard pressure and temperature, V is the actual volume as measured by the experiment, P is the barometric pressure indicated by the barometer and T is the observed temperature of the water bath in degrees Kelvin. Record the oxygen consumption as corrected for standard pressure and temperature.

"As the final step weigh the animal in grams. Add the corrected volumes of oxygen consumed during the six intervals to get the total consumption for one hour and divide the total consumption in cubic centimeters by the weight of the animal in grams to determine the consumption of oxygen per hour per gram of the animal's weight at standard pressure and temperature. A graph can be drawn to show the oxygen consumption of each experimental animal during the experiment by plotting the oxygen consumption, in cubic centimeters, against time in minutes.

"Many variations of the above experiment suggest themselves. One can compare the performance of a single large animal with that of several smaller animals of the same total weight. Similarly, the effects of increased or reduced amounts of light can be measured, but in this experiment take care to maintain the water bath at constant temperature. One animal of a pair can be restrained in a cheesecloth bag while its companion remains free in a separate bottle, thus demonstrating the effects of reduced activity on metabolism.

"Most invertebrates exhibit a decrease in metabolic rate with reduced temperature. To lower the animal's temperature immerse one bottle in an ice bath. If aquatic animals are used, each animal chamber and the thermobarometer should contain a standard amount of the water in which the animal normally lives. The chambers should not be more than half filled with water, however, because the air above the water must contain enough oxygen to supply the animal normally during the experimental run.

"For animals as large as a mouse the equipment that has been described works nicely if the experiment is limited to about 10 minutes. The animal chamber and the manometer tube must be of appropriate size or the mouse will use up all the oxygen and suffocate. Watch the movement of the soap film carefully during the experiment. If the movement of the film slows appreciably, the animal may be running out of oxygen. In this event remove the bottle from the water bath and open it immediately. Then substitute a manometer tube of about twice the diameter of the first one and try again.

"It is also easy to construct a scaled-down version of the spirometer, the instrument used for determining the metabolic rate of humans and other large mammals. The scaled-down instrument [see Figure 1] consists essentially of an airtight animal chamber connected by tubing to a reservoir that is in turn linked mechanically to the stylus of a kymograph, which automatically draws a graph displaying oxygen consumption against time. Apparatuses of this type can be designed for accommodating animals weighing from 50 to 1,000 grams simply by varying the size of the animal chamber and the oxygen reservoir.

"For approximating the dimensions of the oxygen reservoir a good rule of thumb is to assume that each hour an animal at rest will require about one cubic centimeter of oxygen per gram of body weight. For example, a spirometer that contains 250 cubic centimeters of oxygen would be adequate for a one-hour test of a 250-gram animal. The accuracy of the measurements will suffer if the apparatus is made excessively large, because the animal will then consume only a small fraction of the available oxygen, thus reducing the change in volume of the spirometer and the resulting excursion of the stylus.

"The apparatus has been used for rats, mice and baby chicks. The animal chamber is a wide-mouthed, one-pint Mason jar fitted with a self-sealing lid and screw band. Two 1/4-inch holes were drilled in the metal lid, and a one-inch length of 3/8-inch copper tubing was soldered in place over each hole. One copper nipple is connected by rubber tubing to the oxygen supply; the other one is the exhaust and is fitted with a short sleeve of rubber tubing so that this opening can be sealed by inserting a 19 thermometer. It is extremely important that the system be leak-free. Grease the top of the jar and all rubber-tubing connections lightly with Vaseline, clamp on the oxygen-supply tube and check the apparatus for leaks by immersing it in a tub of water.

"Circles of 1/4-inch-mesh hardware cloth were cut to fit the inside of the animal chamber. During the experiment they rest on top of a layer of soda lime (NaOH + Ca(OH)₂), which acts as the absorbent of the carbon dioxide. The stack of wire disks protects the animal from contact with the corrosive chemical; the mesh must be fine enough so that the animal's feet will not slip through the stack and touch the soda lime. For the oxygen supply I have used a tank or lecture bottle; if such a supply is not at hand, one can have a balloon or an inner tube filled with oxygen at a local chemistry laboratory, hospital or welding shop.

"The oxygen supply is connected to the apparatus by flexible tubing and a T fitting, with the animal chamber on the lower arm and the spirometer on the upper arm. The spirometer is made from two telescoping tin cans, one slightly smaller than the other. The outer can of my apparatus is a liquid-soap container of the type that has a plastic neck and screw cap. The bottom of the can was cut out with a can opener. If one uses a different kind of container, one end is cut out and a 10-millimeter hole is drilled in the other end. This hole, or the neck of the soap can, is fitted with a one-hole rubber stopper. A glass tube extends through the stopper to a point l0 millimeters below the open end of the can. The can is mounted securely by a large clamp attached to a ring stand and is filled with water to approximately 20 millimeters from what is now the top. On the outside of the smaller can (I used a frozen-juice can, which clears the soap can by about three millimeters on all sides) a loop of wire is soldered in place exactly in the center of the bottom. The can is inverted inside the larger one and is suspended from the loop by a thread.

"The thread runs up over two pulleys. The first one is about 25 centimeters above the top of the outer can; the second pulley is at the same level but about 15 centimeters to one side. The thread runs around this pulley and down to a counterbalance equivalent in weight to the inner can. A centimeter scale marked on the side of the inner can is convenient. The thread on the counterbalance side carries a pointer that is cut from a piece of photographic film and is held in position between two knots in the thread. The pointer is brought in contact with the smoked surface of a kymograph drum, the thread having been twisted in such a way that its tendency to untwist will keep the pointer in constant but delicate contact with the drum.

"At the beginning of a run the inside can is moved to its lower position and a pinch clamp is placed between the spirometer and the upper arm of the T fitting. The chamber is charged with fresh soda lime and the animal is sealed inside. With the exhaust tube open, oxygen is first bubbled through water, a step that saturates the gas and also serves to indicate rate of flow, and then is delivered to the chamber. After a five-minute equilibration period the clamp below the spirometer is removed and the spirometer is charged with oxygen by briefly closing the exhaust tube. The inner can should now be in its upper position, but its lower rim must extend into the water so that no room air can leak in to dilute the oxygen. The experimental run is started by simultaneously sealing the exhaust tube and clamping the oxygen-supply tube.

"As the animal uses oxygen the inner can of the spirometer moves down and the pointer writes a rising curve on the kymograph drum [see "The Amateur Scientist," April, 1960]. Pressure in the animal chamber and the spirometer remains at atmospheric level since the inner can of the spirometer is freely movable. If the animal seems easily disturbed by activities in the room, drape a black cloth around the chamber. On a day that is warm but not humid, cooling of the chamber can be effected by dampening the cloth occasionally. It may also be necessary to submerge the entire chamber in a constant-temperature water bath; a sink or a plastic dishpan will serve for this purpose. Temperature fluctuations take place much more slowly in water than in air.

"It is necessary to know the volume of oxygen contained in the spirometer can. The volume can be determined by calculating the volume of a cylinder, as in the previous experiment. An even easier way is to fill the inner can with water and determine the depth in centimeters of a given volume of water. For instance, in my setup 325 cubic centimeters of water filled the can to the 10-centimeter mark. Thus each centimeter on the side of the spirometer can, or each centimeter in the vertical rise of the recording stylus, represents a volume of 32.5 cubic centimeters. This is the spirometer-calibration factor. It is also necessary to know how fast the kymograph is turning, that is, how much time is represented by one centimeter of the base line of the graph. An easy way to get this figure is to mark the exact starting point with an arrow on the kymograph tracing; mark the finish point one hour (or fraction thereof) later.

"In a typical experiment I used weanling male rats divided into three groups of two rats each. The animals always had access to food and water. They were weighed daily, at which time the cages were cleaned. The control group, A, received a standard diet (I used a turkey-brooder ration that was available as a finely ground mash). To the diet of group B thyroid hormone was added at a rate of one crushed five-grain tablet of U.S.P. thyroid per 40 grams of feed. Adequate mixing was assured by placing the diet plus ground thyroid hormone in a large paper bag and shaking it well. Group C rats received standard feed to which was added one crushed 50-milligram tablet of propylthiouracil, an antithyroid drug, per 700 grams of feed. (These drugs are prescription items, available only from a licensed pharmacist I have checked with several pharmacists in my area and have been assured that an amateur biologist could probably obtain enough pills for such an experiment by explaining his need to his physician.)

"After one week on these diets weight differences became apparent. Differences in behavior were also beginning to appear: rats receiving the hyperthyroid diet B were definitely more active and 'jumpy,' whereas rats on diet C seemed sluggish. After two weeks on the experimental diets food was withheld overnight, and the following morning oxygen consumption was determined for each animal. Each rat was allowed a five-minute equilibration period in the animal chamber, and then oxygen consumption was monitored for 20 minutes. The chamber was washed, dried and charged with fresh soda lime after each run.

"Barometric pressure on the day of the experiment was 701 torr. Other raw data were recorded in a table that showed in its first four columns the diet given the rat, the number of the rat, the rat's weight in grams and the temperature of the animal chamber in degrees Celsius. There followed for each rat four columns. Column 1 gave the measurement from base line to finish point on the kymograph tracing. Column 2 was obtained by multiplying the figures in column 1 by 32.5, the spirometer-calibration factor. Column 3 was obtained by multiplying these figures in turn by 3 (20-minute run times 3 is 60 minutes). Column 4 was derived by dividing by the body weight. To reduce these figures to standard pressure and temperature (column 5) we used the same formula that was employed in the earlier experiment. The plotted data yielded the accompanying graph [Figure 3].

"The hyperthyroid diet B markedly affected metabolic rate; the loss of weight on this diet arises because the rats run off food reserves instead of storing them. The antithyroid diet C did not have such a dramatic effect on weight, but it depressed oxygen consumption. At the conclusion of the experiment all the rats were given a standard diet, and within a week they had returned to normal weight and behavior.

"In retrospect it would have been preferable to conduct all runs at exactly the same temperature. The conversion to standard pressure and temperature corrects for the expansion of oxygen in the spirometer that accompanies a rise in temperature, but it does not take account of another variable: all warm-blooded animals must expend energy, hence oxygen, in order to maintain a constant internal temperature in spite of varying external temperature. To this extent such determinations of basal metabolic rate are not quite basal, and the results should always be stated as cubic centimeters of oxygen consumed per gram per hour at a stated temperature."

Bibliography

PROTEIN METABOLISM. R. B. Fisher. John Wiley & Sons, Inc., 1954.

METABOLISM AND PHYSIOLOGICAL SIGNIFICANCE OF LIPIDS: THE PROCEEDINGS OF THE ADVANCED STUDY COURSE HELD AT CAMBRIDGE SEPTEMBER 1963. Edited by R. M. C. Dawson and Douglas N. Rhodes. John Wiley & Sons Ltd., 1964.

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