Download LAB 4. CELLULAR RESPIRATION and GLUCOSE

CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
LAB 4. CELLULAR RESPIRATION and GLUCOSE MONITORING
REMINDER!!
Bring a calculator to this lab
PRELAB:
Answer the questions on diabetes (p 6
Print off lab 4 Journal Article and read before class
(see web site under Links); the questions regarding this article are to be
answered and handed in at the end of the lab (you may wish to complete these
before class!)
PART 1. INTRODUCTION TO CELLULAR RESPIRATION
Most cellular reactions require a source of chemical energy, ATP. The major source of ATP is the
oxidation of glucose. Glucose oxidation can be broken down into two major phases.
1.
Glycolysis, an anaerobic process that occurs in the cytoplasm of the cell. Oxygen is not
required for glycolysis which occurs in both aerobic and anaerobic organisms. The end
product of glycolysis is pyruvic acid. A small amount of ATP is made during glycolysis.
2.
Aerobic Cellular Respiration only proceeds when oxygen is available and the cell has
enzyme sysytems in place to utilize it. This phase of glucose oxidation, consisting of the
Krebs Cycle and electron transport, occurs in the mitochondria of the cell. A large
amount of ATP is produced in aerobic respiration -- particularly during electron transport.
ANAEROBIC RESPIRATION
-
Fermentation
Some cells carry out glycolysis and then proceed to metabolize pyruvic acid
anaerobically by the process of fermentation. When a yeast cell carries out fermentation
the end products are ethyl alcohol and carbon dioxide. Other cells will produce lactic acid
from pyruvic acid by fermentation. For example, the muscle cells in your body do this
when their oxygen supply is temporarily depleted during a period of strenuous exercise.
In both cases, the further metabolism of pyruvic acid under anaerobic conditions does not
increase the supply of ATP available to the cell. While an anaerobic organism may obtain
enough ATP from glycolysis/fermentation to sustain life activities, an aerobic organism
requires much more ATP to sustain its activities and can only produce this when pyruvic
acid is further metabolized aerobically.
Yeasts are facultative anaerobes. This means that while they carry out normal
respiration when oxygen is available, they also have the ability to obtain energy via the
process of fermentation when oxygen is not available. Under anaerobic conditions yeast
will metabolize glucose as follows:
C 6H 1 2O 6
2C2H5OH
+
glucose
ethanol
+
2CO2
carbon dioxide
+
ENERGY
+
2ATP
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
PURPOSE
1. To demonstrate the fermentation of sugar by yeast.
2. To determine which sugars yeast can ferment and to what extent.
3. To understand the importance of experimental controls
4. To understand what is meant by a controlled variable, independent variable and
dependent variable.
PROCEDURE
Each group of students will set up one fermentation tube and group results will be
compiled.
•
Practice filling your fermentation tubes with 30 ml of tap water following a demonstration.
If not already done, label and colour band them 1 - 12.
•
Dump out all of the water
•
Weigh out 0.5g yeast in a small tared weigh boat.
•
Measure out 30 ml of the stock solution that your group has been assigned into a 50 ml
beaker. Add one drop of 0.1 NaOH to each solution and enough phenol red to make the
solution pink.
NOTE: phenol red is a pH indicator that is yellow in the presence of acid and pink in the
presence of base. Orange indicates neutral pH (pH 7)
•
NOW........ as a class.....take up the 10 weighboats of 0.5g of yeast and on a countdown
add the yeast to every beaker except no. 2 ( no yeast ) Mix thoroughly with a stirring
rod.
•
Now, add each mixture to the appropriate labelled, color coded fermentation tube as
follows.
•
Tube 1: Glucose + yeast
Tube 7: Galactose + yeast
Tube 2: Glucose (no yeast)
Tube 8: Fructose + yeast
Tube 3: Sucrose + yeast
Tube 9: Sorbitol + yeast
Tube 4: Maltose + yeast
Tube 10: Aspartame + yeast
Tube 5: Lactose + yeast
Tube 11: Sucralose + yeast
Tube 6: Glucose + yeast + NaF
Tube 12: Distilled water + yeast
0
Immediately place all tubes on a tray and place it in the incubator at 37 C for about 20
minutes.
(While the tubes are incubating, start part 2, Glucose Monitoring on page 6)
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
•
After 20 min. observe the tubes in the incubator and note if gas is appearing anywhere. If
any tubes have completely blown; remove the tray and tubes. If not, re-incubate for a
further 20 minutes or until the first tube blows!
•
Remove the tubes for analysis. Measure the heights of the gas columns in each tube and
record these data in the table provided.
RESULTS
Table 1. Fermentation of various sugars and sugar substitutes by yeast
Tube No.
sugar or
substitute
Fermentation Tube Results
yeast
NaF
(inhibitor)
final
colour
gas column
ht. (mm)
1
2
3
4
5
6
7
8
9
10
11
12
1. Although these data are probably best presented as a table; the results could be also be
presented as a graph. What type of figure could you use to present these results?
______________________________
2. What are the products of
aerobic cellular respiration: ________________________________________________
anaerobic fermentation in yeast cells _________________________________________
anaerobic fermentation in muscle cells ________________________________________
3. What gas was produced and measured in this experiment?
___________________
4. Phenol red is a pH indicator that turns yellow in the presence of an acid. What acid is
produced when carbon dioxide is dissolved in water? ______________________
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5. What was the purpose of tubes 2 and 11?_
T2________________________________________________________________
T12_______________________________________________________________
6. Did you get a reaction in either of these tubes? Why or why not?
T2________________________________________________________________
T12_______________________________________________________________
7. Which of the monosacccharide solutions tested
reacted the most?
___________________________
8. List the 3 disaccharides used in the experiment and indicate their component
monosaccharides.
a)__________________
_______________________
_______________________
b)__________________
_______________________
_______________________
c) __________________
_______________________
_______________________
9. Which of the disacccharide solutions above
reacted the most?
_______________________________
10. What disaccharidase (enzyme that breaks
the bond between the 2 monosaccharides
in a disaccharide) is lacking in yeast cells?
________________________________
11. What is sorbitol and did it react? Why or why not?
_____________________________________________________________________
12. What is aspartame and did it react? Why or why not?
______________________________________________________________________
13. What is sucralose and did it react? Why or why not?
______________________________________________________________________
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
14. NaF (sodium fluoride) is a non-competitive inhibitor of the enzyme enolase.
Enolase catalyzes the last step in glycolysis (conversion of phosphoenol pyruvate to
pyruvate). Explain your observations in the tube containing NaF.
___________________________________________________________________
___________________________________________________________________
___________________________________________________________________
15. If an experiment were to examine the effect of various concentrations of NaF on CO2
production by yeast
a)
What would be the independent variable?______________________________
b)
What would be the dependent variable? _______________________________
16. Describe 4 controlled variables that had to be considered in the experiment the class did:
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
______________________________________________________________________
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
PART 2: GLUCOSE MONITORING
INTRODUCTION
Most of the glucose in the body is obtained from dietary carbohydrates and is stored as glycogen
in the liver and skeletal muscles. Glucose is metabolized by the process of cellular respiration to
provide energy in the form of ATP for cells. Insulin and glucagon, two hormones from the
pancreas, help control blood glucose levels. Insulin is needed for cellular membrane permeability
to glucose and for transport of glucose into cells. Without insulin, glucose cannot enter most cells
and remains in the blood. Glucagon stimulates glycogenolysis (conversion of stored glycogen to
glucose) in the liver.
Decreased blood sugar (hypoglycemia) results from inadequate food intake or too much insulin.
When elevated blood sugar (hyperglycemia) occurs, there is not enough insulin or the cells are
unresponsive to insulin; this condition is known as diabetes mellitus. A fasting blood sugar of
greater than 125 mg/dl or 7 mmol/L usually indicates diabetes. To confirm this when the blood
sugar level is borderline or slightly elevated, a fasting (preprandial) blood sugar test and /or a
glucose tolerance test (GTT) is performed.
Symptoms of hypoglycemia could include: slurred speech, headache, tingling of lips, cool sweating, rapid
heartbeat, disorientation, weakness, dizziness, fainting, hunger, nervousness, irritability or tremors.
Causes of hypoglycemia include: insulin excess, cancer (stomach, liver, lung) adrenal gland hypofunction,
malnutrition, alcoholism, cirrhosis of the liver, strenuous exercise, hemolytic disease.
Symptoms of hyperglycemia could include fatigue, dry mouth and skin, increased thirst or hunger, blurry
vision, unexpected weight loss, increased urination, rapid deep breathing or a fruity smell to the breath
(evidence of ketosis).
Causes of hyperglycemia include: diabetes mellitus, diabetic acidosis, adrenal gland hyperfunction
(Cushing’s syndrome), acute myocardial infarction, stress, renal failure, hypothermia, acute pancreatitis,
cancer of the pancreas, congestive
PURPOSE
1. To measure the range of blood sugar levels in the class
2. To understand what diabetes is, and how type I and type II diabetes differ in etiology
3. To understand the clinical significance of the various tests for diabetes
Questions:
1. Explain the cause of hyperglycemia in type I diabetes and in type II diabetes:
2. What is the incidence
of type I diabetes in Canada? ______________
of type II diabetes in Canada? ______________
3. List 4 risk factors for development of type II diabetes:
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
BLOOD GLUCOSE TESTING
A simple blood glucose test will be performed on individuals in the class using the Lifescan
glucometers provided. This is strictly voluntary; if you do not wish to prick your finger or you
have been potentially exposed to any disease transmissible by blood DO NOT DO THE
BLOOD GLUCOSE TEST. Only YOU may prick your finger and handle your test strip.
SAFETY PRECAUTIONS!!
•
Disposable items such as lancets, cotton balls, alcohol swabs, etc.) are to be placed in
marked containers or autoclave bags as directed.
•
You must prick your own finger and only you should handle anything contaminated
with your blood
•
Benches must be washed with bleach solution at the end of the lab.
Procedure
Step #1
preparing the glucometer
•
•
Step # 2
•
•
•
•
•
•
Step #3
follow instructions provided for calibrating the glucometer
place a test strip in the glucometer
preparing to prick finger
GET A BANDAID; OPEN IT UP AND PLACE IT ON A PIECE OF PAPER TOWEL
get a sterile lance from the box (a plastic cap will be over the needle; if the cap
is not in place or it is loose, the needle is not sterile and should be disposed of
in the sharps container!)
get an alcohol swab
insert the lance (needle) all the way into the auto-lance device
twist the cap off the lance
clean the end of the auto-lance device with alcohol!
getting blood
•
•
•
•
•
•
•
Step #4
th
swab your little finger (5 phalanx) with alcohol
press the tip of the finger into the depression at the end of the auto-lance
press the button…..you are now bleeding (exercise caution!)
hold the glucometer and draw blood into the test strip touching the drop of blood to
the end of the test strip (the test strip must remain inserted in the glucometer) by
BANDAGE YOUR FINGER NOW!
read the result.....in approximately 30 seconds.....in mmol/L
record your blood glucose value in the results section
lancet removal / clean-up
•
•
•
•
remove the cap from the lance device, and remove the lancet (needle)
clean the end of the lance device again (using the alcohol swab
place the used lancet in the proper “sharps” container indicated by your instructor.
LEAVING A CONTAMINATED NEEDLE IN THE LANCE DEVICE OR ON THE
BENCH IS EXTREMELY IRRESPONSIBLE AND POTENTIALLY DANGEROUS
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
RESULTS, QUESTIONS, DISCUSSION:
1.
Blood Glucose Results:
No.
(normal post-prandial range is 3.5 – 7.0 mmol/L)
time since last eating
(hours)
blood glucose
(mmol/L)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
2.
What is the range of the blood glucose levels?
___________________
3.
Did anyone fall outside the normal range?
___________________
4.
What is the mean blood glucose level in the class?
___________________
5.
What is the dependent variable? _________________________________
6.
What is the independent variable? ________________________________
7.
Sort the data into 3 groups: ate less than an hour ago, ate 1-3 hours ago, ate more than 3
hours ago. Calculate the mean blood glucose level for each of the 3 groups and the
standard deviation.
Plot these data as a histogram.
group
values
mean
< 1 hr ago
______________________________
________
> 1 hr but < 3 hr
______________________________
________
> 3 hr ago
______________________________
________
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
8.
Is any relationship evident? Why or why not?
______________________________________________________________________
______________________________________________________________________
9.
What hormone is released in the fasted state?
________________________________
What effect does this hormone have on blood glucose levels?
_______
____________
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
GLUCOSE TOLERANCE TEST (GTT)
The standard test for diabetes is a glucose tolerance test. The individual is asked to fast
overnight. In the morning, a fasting (pre-prandial) blood glucose level is recorded. The individual
then consumes a very concentrated glucose solution (containing 75 gm glucose), and blood
glucose levels are sampled at several intervals for the next 3 hours.
Compare the results of a normal and an abnormal GTT as shown below.
Table 1. Results of a GTT in 2 patients
blood glucose concentration (mmol/L)
0 min
30 min
1 hr
2hr
3hr
patient #1
4.7
7.8
8.4
5.6
4.5
patient #2
8.4
12.3
13.7
14.3
14.5
Graph these results below.:
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
QUESTIONS:
1.
How does the fasting blood glucose level differ in these 2 patients?
________________________________________________________
2.
Which of these patients is diabetic? ____________________________
3.
How long does it take for blood glucose levels to peak in a non-diabetic after ingestion of
glucose?
________________________________________________________
4.
How quickly does the normal individual’s blood glucose return to baseline levels?
________________________________________________________
5.
How quickly does the diabetic’s blood glucose return to baseline levels?
___________________________________________________________________
6.
When blood glucose levels drop back to baseline levels, where has the glucose gone?
____________________________________________________________________
7.
What does an abnormal GTT usually signify?__________________________________
______________________________________________________________________
8.
What is the usual reason why blood sugar is elevated?___________________________
______________________________________________________________________
9.
Would the GTT differentiate between type I and type II diabetics? Explain.
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
PART 3. CARBOHYDRATE AS FUEL FOR ENDURANCE EXERCISE
Depletion of muscle and liver glycogen stores, with resulting hypoglycemia, is thought to be a key
factor in fatigue during prolonged exercise. Total carbohydrate stores in the body are limited and
have been estimated to last for approximately 70 - 90 minutes when exercising continuously at
65-70% VO2max. It has been shown that consuming carbohydrates during endurance events
that exceed 60 minutes duration prolongs time to fatigue and improves performance.
Many carbohydrate supplements have been developed (and marketed) for use during endurance
exercise. In the development of these supplements, considerations have included:
•
type of carbohydrate (which monosaccharides, disaccharides, polysaccharides and in
what combinations)
•
how quickly the carbohydrate is absorbed
•
optimal concentration of carbohydrate
•
digestibility (tolerance of the user)
•
palatability (taste, texture, ease of consumption)
Purpose:
•
review a study related to carbohydrate metabolism in the context of exercise
•
apply knowledge of glucose metabolism (cellular respiration)
•
apply knowledge of sugar, water and sodium absorption from the small intestine
•
apply knowledge of normal vs. abnormal glucose levels
•
interpret experimental results
•
examine ingredients of carbohydrate supplements marketed for endurance exercise
Procedure
1. Examine the exercise fuels / supplements and complete the table on the following
page
2. Having read the journal article provided (found under links on Peggy’s web site) and
answer the questions that follow.
Jentjens R., Venables M.C., and Jeukendrup A.E. Oxidation of exogenous glucose,
sucrose, and maltose during prolonged cycling exercise J. Applied Physiology 96: 12851291, 2004
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
Examine the carbohydrate products displayed on the bench. These products are
marketed for consumption during endurance exercise. Complete the following table for
6 of the products.
product
carbohydrate
sources
indicate if each
of these is a
mono, di or
polysaccharide
any noncarbohydrate
energy
sources or
any caffeine in
product?
Na+
K+
Questions:
1. Why are Na+ and K+ usually added to products such as these?
2. Based on the results of the Jentjens et al. study, which product(s) would you recommend to a
triathlete as fuel during a triathlon?
3. Which product would you choose to use yourself during an endurance event and why?
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
JOURNAL ARTICLE QUESTIONS:
1. What is CHO?
________________________
2. What are the products of CHO oxidation in the body? ___________________________
3. Which of these products was measured to evaluate rate of CHO oxidation
in the cyclists? ________
4. What is exogenous CHO and what is endogenous CHO in the context of this study (be
specific; specifically what are the endogenous sources of CHO?)
5. Describe 4 controlled variables in this experiment (see methods):
6. How long did the cyclists exercise for? ___________
At what intensity? ___________
7. What sugars (or combination of sugars) were tested as energy sources?
8. What solution was the experimental control?
____________________________
9. What 2 disaccharides were used and what are the monosaccharide components of these
disaccharides?
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CAMOSUN COLLEGE BIOLOGY 144 (2010) LABS
10. Over the first 15 minutes of exercise (Fig 3A) plasma glucose rises except in subjects
who consumed water. Why?
11. Did plasma glucose levels fall significantly over the duration of the experiment (compared
to plasma glucose levels at time 0)?
12. Why do plasma lactate levels rise in the first 15 minutes (Fig 3B)?
13. Which sugar or mixture of sugars provided the most readily available energy to the
athletes (as evidenced by exogenous CHO oxidation rates)?
14. What explanation do the authors suggest for the observation above?
15. The authors note that the addition of the electrolyte Na+ aids in gastric emptying and
intestinal absorption of glucose. Why?
16. What was the concentration of NaCl in the solutions given to the cyclists? ___________
17. Did the drinks supply K+ in any form? _______________
18. Which carbohydrate solution was associated with the
fewest gastrointestinal complaints?
_________________________
19. Based on the results of this experiment, can the authors conclude that a mixture of
different sugars will enhance performance of cyclists? Explain.
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