STUDENT INVESTIGATION 4.2

Background

The measurement of glomerular filtration rate (GFR) in space is one of the very important measurements to make in order to understand how the body responds to the "upward" fluid shift that occurs in space. In fact, it is one of the major components of Dr. Leach's space flight experiment. The technique that is used to measure GFR is known as the plasma clearance method. The term "plasma clearance" is somewhat self-explanatory; it is used to express the ability of the kidneys to clean, or "clear," the plasma of various substances. This technique is essentially the same as the "dilution method" that we discussed in the previous chapter because it involves the determination of how fast the body is able to eliminate a chemical tracer, or "marker" substance from the bloodstream. The difference between the "plasma clearance method" and the "dilution method" is that the tracer substance that is chosen for the plasma clearance method will be filtered out of the bloodstream and eliminated in the urine. The tracer that is chosen to carry out the dilution method is selected for its ability to stay in the bloodstream and not be eliminated by the kidneys.

Specifically, the plasma clearance method to measure GFR is based on a determination of how quickly a chemical tracer that is injected into the bloodstream of the astronauts is removed from the blood plasma by filtration in the glomeruli of the kidneys. Thus, the method involves determining the rate of loss of the tracer.

The tracer used in this technique must have the following properties:

1. It should be non-toxic to the human body;
2. It should be able to be measured accurately by chemical or physical means;
3. It should be freely and evenly diffusible throughout the extracellular fluid (ECF) compartment;
4. It should be removed from the blood only by filtration in the kidneys;
5. It should not be reabsorbed into the blood from the renal tubules or other parts of the urinary system;
6. It should be completely excluded from the intracellular fluid (ICF) compartment.

Figure 9. For this demonstration activity you are simulating, with each successive dilution, the kidney's "clearance" of a tracer out of the bloodstream. After the addition of methyl violet indicator, test tube # i win represent the initial concentration of the tracer that is injected into the bloodstream. Test tubes #2, #3, #4, #5, and #6 will represent the tracer concentration in the bloodstream after longer and longer periods of glomerular filtration.

First of all, you will be using a concentrated acid for this experiment. Therefore, review the laboratory safety instructions with your teacher. Above all, use safety goggles and be aware of the chemical wash facilities (at least the sink) in the event that you splash the acid onto your skin. Secondly, when mixing acid and water, always remember: never add water to acid. This will cause a violent and dangerous reaction. For this experiment, you will only add acid to water.

For this activity, you will be diluting acid/water solutions five different times. As an analogy, think of the water in each test tube as your blood supply and think of the concentrated hydrochloric acid (HCL) as the tracer substance that will be injected into your bloodstream. Each time you remove some of the solution from one of the test tubes, you should imagine this as the process of glomerular filtration where some of the tracer substance is being eliminated from the bloodstream. Finally, you will add a special indicator substance to each of the completed HCL/water solutions so that you are able to analyze the relative differences in HCL concentration of the six solutions. This will give you an idea of how different tracer concentrations determined from blood samples taken from the astronauts will indicate the rate at which the tracer is being eliminated from the system.

Remember, analogies can only serve as a partial representation of what is really happening. It is important to remember that for the actual determination of glomerular filtration rate, the time between blood samples must be carefully recorded. You will be asked to think about why and to respond to a question about this at the end of the experiment. You may want to discuss this with your experiment group.

Finally, since this is a demonstration experiment, there is no need to develop an hypothesis about the results that you expect. There are, however, a few objectives for you to keep in mind that will define what you are supposed to learn from this activity. From your experiment, you should be able to:

1. perform a serial dilution;
2. understand that indicator molecules respond to a different tracer concentration;
3. realize that color indicators are an accurate method of measuring concentration differences; and, probably most important
4. translate how an analogy (in case, the serial dilution) is similar to and different from reality (in this case, a determination of GFR).

Materials

Safety goggles

Concentrated HCL acid

Methyl violet indicator

Six test tubes

Graduated cylinders: 10 ml and 100 ml

Distilled water

Cork

1. Label six test tubes 1, 2, 3, 4, 5, and 6.
2. Place 25 ml of distilled water into each test tube.
3. Measure 10 ml of concentrated HCL acid and add to test tube #1. Cork the top of the test tube and shake it.
4. Remove 10 ml from test tube #1 and add it to test tube #2. Cork and shake.
5. Remove 10 ml from test tube # 2 and add it to test tube # 3. Cork and shake.
6. Repeat procedure for test tubes #4, #5, and #6.
7. Remove 10 ml from test tube #6 and discard.
8. When complete, each test tube will contain 25 ml of fluid.
9. Place 2 to 3 drops of methyl violet indicator to each test tube.
10. Observe and record the final colors of each solution. It may be necessary to "rate" the colors numerically (for instance, between 1 and 12 to obtain values that reflect the relative color differences among the test tubes).

1. What is an indicator and how does it work?
2. What general term is used in place of "indicator" for the substance used in the plasma clearance technique described for the measurement of GFR? And what is the name of the chemical product used for this purpose?
3. How is the indicator used in this activity similar to and different from the chemical product used to measure GFR?
4. How would you design a test that is more similar to the actual measurement technique that is used to measure GFR? Hint: Time.