LAB 8

                                     THE EFFECT OF THYROXIN ON RESPIRATORY RATE

I. Introduction

The hormone Thyroxine (T₄) and Triiodothyronine (T₃) are produced by the thyroid gland and affects both the growth characteristics and general metabolism of an animal.

Thyroxine (T₄)                                           Triiodothyronine (T₃)

The purpose of this exercise is to investigate the role that the thyroid hormones play in general oxidative metabolism of the rat. We will do this by measuring the oxygen consumption of three groups of rats. We may use one or two different procedures to measure the basal metabolic rate (BMR) of normal, hyperthyroid and hypothyroid rats as reflected in their oxygen consumption. Three groups of three male rats (approximately 150 grams each) will be measured.

            1*         A control group to which no hormone or other drug has been given.

            2*         A hypothyroid group established by the oral administration of thiouracil (a goitrogenic     agent) for two weeks prior to the lab.

            3*         A  temporarily induced hyperthyroid condition brought about by the intra-peritoneal         injection of thyroxine.

Hyperthyroidism is the presence of excess amounts of the hormone thyroxine and can be induced in experimental animals by the oral administration of thyroxin in the food, or by injection of 25 ug thyroxine  intraperitoneally every thrid day for 2 weeks. We are using the later procedure.

Hypothyroidism can be induced by destruction or surgical removal of the thyroid gland. But it is more convenient to destroy the thyroid by the administration of thiouracil in the drinking water of the animal for a two week period prior to the scheduled lab (0.1% solution) or by mixing with the food in the ratio of 1 gm of thiouracil per kg of food.

Thiouracil

Thiouracil is a goitrogen, a chemical substance which decreases thyroxin production and has the advantage that it will not affect the parathyroid gland as would surgical removal or destruction.

II. Objectives

            l.          Measure the rate of 0₂ consumption and CO₂ production in the rats

            2.         Calculate the Basal Metabolic Rate (if using traditional respirometers)

            3.         Characterize the effects of altered thyroid activity

III. Procedure

There are two methods we have available for characterizing oxygen consumption, and one for the characterization for CO₂ production. The first involves the use of a traditional respirometers fashioned from vacuum desiccators. Soda Lime (made up of NaOH and Calcium) is added to the chamber to absorb C0₂.

Since the soda lime absorbs C0₂, any decrease in volume of the enclosed system represents the amount of 0₂ utilized by the animal. In the system we use we will continuously add a measured volume of air via a syringe and track the time it requires for the animal to utilize the oxygen within the injected air is measured (see figure 1).

Figure 1. Traditional respirometer setup

The benefit of this experimental system is that it gives a direct measure of oxygen utilized. Oxygen utilization is a direct measure of the metabolism of the animal and by adjusting for animal weight, we can have an accurate measure BMR.

Unfortunately, there are several problems with these systems. First, the soda lime is expensive and its difficult to determine how fresh, or efficient it is in absorbing the CO₂. Second, the system relies on a very tight seal of the chamber and all the fittings, any air leak will invalidate the results.

The second procedure we have available is to use gas sensors which directly measure changes in oxygen and carbon dioxide levels (see figure 2). The sensors send their output to the chart software of the PowerLab system as a graphical readout. This would seem to be an ideal setup, but it also has its limitations. The major limitation is there is no absolute value or measurement of the amount of oxygen utilized, or carbon dioxide generated.

One way to get around the limitations of the meters is to try to calibrate them, then see if we can apply the calibration to the output we get with the animals. This is a procedure that can be used for the probe calibration.

1.      Calibrate the CO₂ probe by having the reading of atmospheric gasses as representing “Zero”, as the atmospheric CO₂ concentration is well below 1%. The sensor should then be exposed to a known concentration of CO2. The easiest way to do this is to collect a sample of air from our CO2 incubator in the microbiology lab which is set to have a 5% CO₂ atmosphere.

2.      To calibrate the O₂ sensor, sample 100% N₂ which should equate to a reading of 0% O2. Then sample the atmosphere, which should give a value of 21% O2.

Figure 2. CO₂ and O₂ gas sensors in animal chamber

Here are the steps for the experiment.

1.         Record the weight of your experimental subjects to the nearest gram.

2.         Calibrate the sensors if necessary (see above)

3.         Setup the “Chart” software settings. Speed – 100, 5 Volts, Autoscale, 5 min is a good place to    start to dial in the settings for the sensors. 

4.         Remove the top of the chambers and place the rat inside. Make sure that you can read the thermometer in the chamber.

5.         Repeat the steps above three times, allowing 5 minutes between runs to recharge the chamber     with fresh air and re-equilibrate the sensors. Determine the average milliliters of oxygen used per       minute for your rat.

6.         Repeat steps for each of the experimental conditions (Control, Hypothyroid, Hyperthyroid),

IV. Questions/Data Collection

One of the advantages of using a traditional respiometer system is that you can directly measure the volume of oxygen utilized. The oxygen probes we will be using are easy to use, but it is more challenging to calculate that absolute value of oxygen used, which is why we will try to calibrate the probes (see above). Even if we cannot get absolute values of oxygen consumed, we should be able to compare differences in gas utilization between the control and experimental groups.

1.         What are the differences in powerlab readouts for the experimental and control animals for both CO₂ and O₂?

2.         What are the significances of these differences?  Include a comparison of the rates for hyperthyroidism, hypothyroidism and control, with specific reference to the action of thyroxine

3.         If the data are not what you would have predicted, why do you think you got the data you did?

4.         Use Excel or Sigmaplot to come up with a graphical representation summarizing your data.

                                    Control                            Hyperthyroid                          Hypothyroid

V. Materials:

1.         10 male rats (about 150 gms) and cages, rats must be obtained, acclimatized, 3 of the 9 are treated with a Thiouracil two weeks prior to the lab

2.         Traditional vacuum chambers and Manometer systems (used last time)

3.         O₂ and CO₂ probes with animal chambers

4.         PowerLab Systems

5.         Thyroxine and Syringes

6.         Barometer(s)

7.         Thermometers

8.         Soda Lime (if available, do not order or buy if not on hand)

9.         Calculators

CAUTION: THIOURACIL IS EXTREMELY TOXIC AND WILL DESTROY YOUR THYROID AS WELL AS THAT OF THE EXPERIMENTAL ANIMAL - USE WITH EXTREME CAUTION! DO NOT BREATHE THE DUST WHEN MAKING UP THE SOLUTION!

VI. References:

Grinnell, A. and Barber, A.A. 1976. Laboratory Experiments in Physiology, Ninth Edition, Mosby Publishers.

Tharp, G.D. Experiments in Physiology. 1980. Experiments in Physiology, Fourth Edition, Burgess Publishers.