Lab 6 – Cellular Pathology
Objectives



Compare and analyze temperature effects on lactase activity
Compare and analyze pH effects on catalase activity
Evaluate blood and urine glucose levels to determine diabetic patient
Introduction
Many diseases and ailments are the result of chemical malfunctions at the cellular level.
Almost every one of us knows someone who is either lactose intolerant or diabetic. In both
cases, proteins play a central role. For lactose intolerance, enzymatic proteins are absent. For
diabetes, the protein in question is insulin. Both of these disorders illustrate the importance of
proteins in a living system.
Enzymes are proteins that act as biological catalysts by speeding up the rate of chemical
reactions that would otherwise take extensive periods of time to occur. By lowering the energy
of activation of chemical reactions, enzymes thus speed up the rate of such reactions. For
example, the enzyme catalase breaks down hydrogen peroxide, a highly reactive and dangerous
end product of several metabolic reactions, into oxygen and water. The presence of catalase
allows this reaction to occur about nine and half BILLION times faster than the uncatalyzed
reaction. Enzymes are involved in almost every aspect of life, and they often work together in
tandem to form complex degradation or synthetic pathways, known as metabolic pathways.
These metabolic pathways are involved in such functions as the breakdown of ingested food to
provide energy for the cell and the synthesis of new cellular materials, such as lipids and
carbohydrates.
Though enzymes are a specialized class of proteins, they still share the same
characteristics of all proteins. They have a primary, secondary, tertiary, and quaternary structure.
Because each enzyme has a unique structure, each enzyme tends to be very specific with respect
to the reactions that it speeds up. Most enzymes catalyze only a single reaction, although a few
are able to catalyze multiple reactions that are similar in nature. Substrate is the term used to
refer to the molecule or molecules that the enzyme acts upon. The substrate or substrates bind
tightly to specific regions of the enzyme known as active sites. Once a reaction occurs, the
enzyme, unchanged by the process, reassumes its original shape and awaits the arrival of more
substrate(s).
The three-dimensional shape of an enzyme is critical to its proper functioning. If this
shape is altered, the enzyme will work less efficiently or, in cases of extreme alteration, cease
functioning altogether. The bonds that maintain the shape of the enzyme are easily disrupted by
heat. If the temperature increases too much, the bonds will break, and the enzyme will unfold
and become inactive. This is known as heat denaturation. Enzyme activity may be altered also
by changes in pH, substrate concentration, enzyme concentration, or inhibitors.
In lab, you will be examining the effects of temperature on the enzyme lactase. This
enzyme is present in the small intestine and is responsible for breaking down the sugar lactose
into the simple sugars glucose and galactose. People deficient in this enzyme are lactose
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Lab 6 – Cellular Pathology
intolerant and are unable to digest dairy products, a common source of lactose. In addition, you
will also observe how pH affects catalase activity.
While lactose intolerance can cause a great deal of discomfort for its sufferers, diabetes
can result in death. Approximately 25.8 million people of all ages in the U.S. have diabetes.
This means that about 8.3 % of the U.S. population have diabetes. Of the 25.8 million, 7.0
million go undiagnosed. (from http://diabetes.niddk.nih.gov/DM/PUBS/statistics/)
What Is Diabetes?
Diabetes is a disorder of metabolism--the way our bodies use digested food for growth
and energy. Our digestive enzymes break down most of the food we eat into a simple sugar
called glucose. Glucose is the main source of fuel for the body.
After digestion, the glucose passes into our bloodstream where it is available for body
cells to use for growth and energy. For the glucose to get into the cells, insulin must be present.
Much like enzymes, insulin is a specialized protein called a hormone. The pancreas produces
insulin. When we eat, the pancreas automatically produces the right amount of insulin to move
the glucose from our blood into our cells. In people with diabetes, however, the pancreas either
produces little or no insulin, or the body cells do not respond to the insulin that is produced. As a
result, glucose builds up in the blood, overflows into the urine, and passes out of the body.
Diabetes mellitus translates to “honey sweet passing through.” The body loses its main source of
fuel even though the blood contains large amounts of glucose.
The three main types of diabetes are:
 Immune-mediated diabetes mellitus or Type I diabetes mellitus
 Type II diabetes mellitus
 Gestational diabetes
Type I diabetes is considered an autoimmune disease. An autoimmune disease results when the
body's system for fighting infection (the immune system) turns against a part of the body. In Type I
diabetes, the immune system attacks the insulin-producing beta cells in the pancreas and destroys
them. The pancreas then produces little or no insulin.
Someone with Type I diabetes needs daily injections of insulin to live. At present, scientists do
not know exactly what causes the body's immune system to attack the beta cells, but they believe that
both genetic factors and viruses are involved. Type I diabetes accounts for about 5 to 10 percent of
diagnosed diabetics in the United States. Symptoms include increased thirst and urination, constant
hunger, weight loss, blurred vision, and extreme tiredness. If not diagnosed and treated with insulin, a
person can develop ketoacidosis, which can bring about a life-threatening coma. Type I diabetes
develops most often in children and young adults, but the disorder can appear at any age. Symptoms
of Type I diabetes usually develop over a short period, although beta cell destruction can begin
months, even years earlier.
Type II diabetes is the most common form of diabetes. About 90 to 95 percent of people with
diabetes have Type II. This form of diabetes usually develops in adults over the age of 40 and is most
common among adults over age 55. Unfortunately, even young children are developing Type II
diabetes. About 80 percent of people with Type II are overweight. In Type II, the pancreas usually
produces insulin, but for some reason the body cannot use the insulin effectively. The end result is the
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Lab 6 – Cellular Pathology
same as for Type II diabetes: an unhealthy buildup of glucose in the blood and an inability of the body
to make efficient use of its main source of fuel. The symptoms of Type II develop gradually and are
not as noticeable as in Type I diabetes. Symptoms include feeling tired or ill, frequent urination
(especially at night), unusual thirst, weight loss, blurred vision, frequent infections, and slow healing
of sores.
Gestational diabetes develops or is discovered during pregnancy. Gestational diabetes results
when hormones from the placenta block the normal action of insulin. This type usually disappears
when the pregnancy is over, but women who have had gestational diabetes have a greater risk of
developing Type II later in their lives.
Glucose Tolerance Test
In diagnosing diabetes, several tests are used to determine what metabolic error is causing
the disease. The glucose tolerance test assays the ability of the body (especially the pancreas) to
respond to excess ingestion of glucose. The changes of blood glucose level following glucose
ingestion (1 g/kg body weight) are markedly different from the normal and diabetic person. This
difference is shown in Figure 1. In a normal person, the blood glucose level rises from 90 mg/dL
to around 140 mg/dL in 1 hour and falls back to normal within 3 hours or even below due to
excess insulin release by the pancreas. A diabetic shows a hyperglycemic response in which the
blood glucose level rises from about 120 to 160 mg/dL to as high as 300 mg/dL and then slowly
falls to the fasting diabetic level after 5 to 6 hours. A diabetic’s abnormal response is caused by
the inability of the pancreas to secrete additional insulin in response to elevated blood glucose
levels.
For the glucose tolerance test, the patient should report to lab in a fasted state (not having
eaten for the last 12-18 hours). The patient’s blood glucose levels are determined using a
glucometer. The patient then drinks a commercial flavored solution of 25 % glucose. After
ingesting the glucose solution, blood glucose levels are tested at 30 min., 60 min., 90 min., 120
min., 180 min., 240 min., and 300 min.
Figure 1. Typical glucose tolerance test data.
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Lab 6 – Cellular Pathology
6.1 Lactase activity
1.
2.
3.
4.
5.
6.
7.
8.
9.
Enzymes affect the amount of products formed per unit of time. Lactase breaks the
disaccharide lactose into glucose and galactose. In lab, we will measure the product
glucose.
Write your predictions about glucose production on the QUESTIONS page.
Pipette 5 mL of milk into each of four clean test tubes.
Place one test tube into each of the four different temperatures (0 0C, 25 0C, 37 0C, and
100 0C). Make sure to identify your group’s test tubes.
0 0C = freezing point
25 0C = room temperature
37 0C = body temperature
100 0C = boiling point
Incubate the test tubes in the appropriate temperatures for 3 minutes before testing the
initial glucose levels and then leave these test tubes in the appropriate temperatures for
the entire experiment.
Reminder: only open the lid of the glucose test strip container when necessary.
Keep the lid on at all times.
At the end of the 3 minutes incubation, dip reagent area of one glucose test strip into the
milk of one test tube. Take out the test strip immediately and wipe any excess off milk
on the lip of the test tube. [Note: if the test tube is too long for you to put glucose strips
inside directly, use a glass stir rod to put milk on the reagent area.]
Wait 30 seconds.
Compare the reagent area color with the chart on the strip container. Record the glucose
levels in mg/dL in Table 1 on QUESTIONS page.
10.
Measure the glucose amount for the other test tubes of milk and record data.
11.
Continue to keep the test tube at the appropriate temperature after the initial 3
minutes incubation.
Add five drops of lactase solution down the middle of each test tube. Try NOT to dribble
the enzyme down the side of the test tube. Gently swirl the test tube to mix the lactase
with the milk.
Allow the enzyme to react for 3 minutes.
12.
13.
14.
After lactase has been reacting with lactose for 3 minutes, check the glucose levels in
each test tube using steps 7-9.
15.
To determine the amount of glucose produced by the enzyme, subtract the initial glucose
reading from the final glucose reading.
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Lab 6 – Cellular Pathology
6.2 Catalase Activity
1.
Since catalase breaks down hydrogen peroxide into oxygen and water, you will be
measuring the amount of oxygen produced in the form of bubbling. Use the method
below for recording your observations.
No bubbling
0
Good bubbling
++
Moderate bubbling
+
Very good bubbling
+++
2.
To practice observing catalase activity, obtain 3 test tubes. Mark each test tube 3 cm
from the bottom.
In each of the three test tubes, fill to the 3 cm mark with hydrogen peroxide.
Add a pinch of sand to one test tube, 1 cubic cm of fresh potato to another test tube, and 1
cubic cm of macerated potato (using mortar and pestle) to the third test tube.
Record any bubbling in Table 2 on QUESTIONS page. [Note: Potato is a good source of
the catalase enzyme.]
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
To observe the effects of pH on catalase activity, obtain 3 more test tubes.
Mark each test tube 3 cm and 4 cm from the bottom.
In each of the three test tubes, fill to the 3 cm mark with hydrogen peroxide.
In one tube, fill to the 4 cm mark with water; in another test tube, fill to the 4 cm mark
with 0.1 M sodium hydroxide; and in the third test tube, fill to the 4 cm mark with 0.1 M
hydrochloric acid (HCl). Mix contents gently by swirling test tubes.
Using pH test strips, measure the pH of each test tube.
Add 1 cubic cm of macerated potato to each of the three test tubes. Wait 1 minute.
Record any bubbling in Table 3 on QUESTIONS page.
6.3 Blood Glucose Tolerance test for two patients
1.
2.
3.
A blood glucose tolerance test was done on two patients already, and their results are
recorded on laminated cards, which are placed in an envelope.
Obtain one laminated card that has values of the blood glucose test for Patient 1 and
Patient 2.
Record these values in Table 4 on QUESTIONS page.
6.4 Urine glucose test for two patients
1.
2.
To measure the urine glucose levels, dip the reagent area of a glucose test strip into
Patient 1’s urine sample and remove the strip immediately. Wipe off any excess urine.
Wait 30 seconds; compare the reagent area color with the chart on the strip container.
Read glucose values in mg/dL and record in Table 5 on QUESTIONS page.
Measure Patient 2’s urine glucose and record.
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Lab 6 – Cellular Pathology
6.1 QUESTIONS
1.
At which temperature (0 0C, 25 0C, 37 0C, or 100 0C?),
a. do you predict glucose production would be highest? Propose an explanation.
b. do you predict glucose production would be the lowest? Propose an explanation.
Complete Table 1 below to show activity of lactase.
Table 1. Levels of glucose produced from lactase.
Temperature
Glucose levels before
lactase (mg/dL)
Glucose levels after
lactase (mg/dL)
Glucose levels
produced from lactase
(mg/dL)
0 0C
25 0C
37 0C
100 0C
2.
Based on your results seen in the third column in Table 1, DISCUSS if your results
support your predictions in question 1 above.
3.
Suppose you had added more lactase to the test tube at 37 0C, how would glucose
production have been affected and explain why.
4.
Suppose you had added more milk to the test tube at 37 0C (but maximum rate of enzyme
activity has not been reached yet), how would glucose production have been affected and
explain why.
5.
Suppose you had changed the pH of milk solution at 37 0C to become pH of 1, how
would glucose production have been affected and explain why.
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Lab 6 – Cellular Pathology
6.
Suppose you had added an enzyme inhibitor to the milk solution at 37 0C, how would
glucose production have been affected and explain why.
7.
Provide two ways in which you could have made an error during the lactase experiment.
6.2 QUESTIONS
Complete Tables 2 and 3 below to show activity of catalase.
Table 2. Catalase activity, without changing pH, to observe what “bubbling” means.
Tube
1
Contents
Bubbling
2
3
Table 3. Catalase activity, with changing pH.
Tube
1
pH
Contents
Bubbling
2
3
8.
Use your results in Table 3 above to explain if changing the pH by adding NaOH or HCl
changed catalase activity.
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Lab 6 – Cellular Pathology
6.3 QUESTIONS
Complete Table 4 below to show blood glucose results for Patients 1 and 2.
Table 4. Blood glucose tolerance test for Patient 1 and Patient 2.
Time (min)
Blood glucose (mg/dL)
Patient 1
Blood glucose (mg/dL)
Patient 2
0
30
60
90
120
180
240
6.4 QUESTIONS
Complete Table 5 below to show urine glucose results for Patients 1 and 2.
Table 5. Urine glucose test for Patient 1 and Patient 2.
Patient 1
Patient 2
Urine glucose (mg/dL)
9.
Based on the blood and urine glucose tests, which patient is diabetic?
10.
Based on the diabetic’s urine glucose test, explain why a diabetic would be really thirsty
quite often.
HAND IN QUESTIONS PAGES (6-8).
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