Lipid Digestion – Lipid Hydrolysis

 

1. Laboratory Objectives:

Students will:

• Become familiar with some of the enzymes and end products in lipid digestion
• Understand how the pH and temperature of an environment affects digestive enzyme function
• Understand how bile salts impact lipid digestion

Laboratory report due:

Procedure Summaries and Data Records & Discussion Questions (Sections E, F & G)

 

B. Introduction

The bulk of enzymatic activity for the digestion of protein and fat occurs in the small intestine. However, there are several minor digestive processes that start in the mouth and stomach.

As acidic chyme leaves the stomach and enters the small intestine, the pancreas is stimulated to release pancreatic juice.  Pancreatic juice contains amylase, different proteases, and lipase along with other components such as bicarbonate.  Additionally, the gall bladder releases bile into the small intestine.  Lipase is the enzyme that hydrolyzes lipids (fats). Bile is used to emulsify dietary fat by increasing the surface area that lipase can act upon. As is true with carbohydrates, protein and fats are also broken down into their smallest units to allow for absorption.

In this lab you will use lipase to study the digestive process that takes place before absorption of fats.  Additionally, you will demonstrate the impact bile salts have on lipid digestion.  You will also determine how changes in temperature and pH may affect the digestive process.

 

Lipid Hydrolysis

The majority of dietary fat consists of triglycerides.  Triglycerides are broken down into glycerol and fatty acids by lipase.

The salivary glands do possess lingual lipase that may initiate digestion of short chain fatty acids in the mouth, but the majority of fat digestion occurs in the small intestine.

Digestion of fats is slightly more complicated than carbohydrate and protein digestion because fats must first be emulsified by bile to increase their surface area and to allow the water-soluble lipases to get to the triglyceride molecules.  Increased surface area makes lipase more effective.

The basis of the assay used in this laboratory exercise is the fact that lipase breaks triglycerides into fatty acids and glycerol.  As the name implies, fatty acids are acids and so have a lower pH.  The heavy cream used in this experiment has a colored compound called litmus added to it.  Litmus is an alkaline solution that turns from blue to pink in the presence of acid.  Therefore, as the pH decreases when lipids are hydrolyzed to fatty acids, the decrease in pH will be visualized by the color change from blue to pink.

 

Review of Factors that affect Enzyme Function

Each enzyme functions best under specific environmental conditions unique to that enzyme. An alteration in conditions such as temperature or pH will affect the ability of the enzyme to function.

 

The pH in the GI tract. 

Some digestive enzymes work in the mouth, some in the stomach, and some in the small intestine. Since there are changes in the environmental conditions as food passes through the digestive tract, the digestive enzymes in various parts of the GI tract have to be specifically suited for the environment in which they are supposed to act.  The mouth has a neutral pH, the stomach is very acidic (pH of ~ 2) and the small intestine has an alkaline pH.

An enzyme that functions in the upper GI tract, like the mouth or stomach, will stop functioning when it is carried along with food to the lower GI tract, where conditions are significantly different.  For example, the enzyme amylase is produced and works in the mouth where pH is close to neutral and the small intestine, where the pH is slightly basic.  When the salivary amylase in the mouth is exposed to the extremely acidic environment of the stomach, the enzyme will become denatured. This means that the structure of the enzyme is permanently disrupted and it will no longer function.

In the body there are naturally occurring substances that help either prevent or minimize a change in pH. These compounds are called buffers.

Temperature in the GI tract

Heat has a tremendous effect on the activity of enzymes. The body is naturally at a temperature of around 98.6F.  If the body were to be colder than its normal temperature, the enzymes would work much more slowly (be less active). If the temperature were to be a little warmer, the enzymes would work a little faster. But, if the temperature were to get very hot, for example, at boiling point, the extreme heat would denature the enzymes (in the same way extreme acid denatures enzymes). This would essentially “kill” the enzyme and it would no longer function.

 

1. Materials Needed

12 test tubes

Black Sharpie pen

2 – 150 ml Beakers

Hot plate

Ice

12 Disposable transfer pipettes (keep dirty pipettes separate from clean)

Test tube rack

Mortar and pestle

Graduated cylinder 10 ml

 

Dropper bottles

1.0 N Hydrochloric acid (HCl)

Buffer

Distilled water

Lipase

 

Supply table

Water bath set at 37^oC

Heavy cream with litmus indicator added

Oil

Food coloring

Bile salts (sodium taurocholate)

 

D. Procedure

 

Digestion of Lipids

A).  Set up

1. Obtain 12 test tubes from your supply tray.
2. Mark the tubes #1-10 with the black Sharpie pen. Also label with a group identifier. Mark the remaining two tubes “A” and “B”.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

B).  Observation of Bile Emulsification

3. To tube A, add 10 drops of water and 1 drop of food coloring.   Then add 3 drops of oil.

 

4. In your mortar and pestle, ground together about 25 drops of oil with a very small pinch of bile salts (3-5 grains of bile salt).
5. To tube B, add 10 drops of water and 1 drop of food coloring. Swirl. Then add 3 drops of oil with bile salts from your mortar.

 

 

6. Swirl tubes A and B and observe how the bile salts affect the oil.

 

7. Record observations on the Lipid Digestion Summary and Data Record.

 

C).  Controls

8. To tube #1, add 12 drops of water and 3 drops of litmus cream.
9. To tube #2, add 12 drops of water and a small pinch of bile salts; no more than 3-5 grains of bile salt.
10. Use your graduated cylinder to obtain about 10 ml of litmus cream from the supply table.
11. In your clean mortar and pestle, ground together about 10 ml of litmus cream with a small pinch of bile salts (this cream will be used in tubes #3, 8, 9, and 10).

 

12. To tube #3, add 12 drops of water and 3 drops of litmus cream with bile salts (mortar).
13. To tube #4, add 12 drops of buffer and 3 drops lipase solution.

 

 

D). Enzymes and emulsifiers in lipid digestion

 

14. Set up a boiling water bath by filling a 150 ml beaker with about 75 ml tap water and placing it on hot plate.
15. Set up an ice bath by filling a 150 ml beaker with about equal parts tap water and ice.
16. To tube #5, add 12 drops of buffer, 3 drops of lipase solution, and add 3 drops of litmus cream (directly from bottle on supply table—no bile salts added).
17. To tube #6, add 12 drops of buffer, 3 drops of lipase solution. Place in a boiling water bath for 5 minutes using a test tube clamp. After 5 minutes, remove the tube.  Then, add 3 drops of litmus cream (directly from bottle—no bile salts).
18. Place tube #7 in the ice bath and add 12 drops of buffer and 3 drops of lipase solution. Let the tube sit for 2 minutes, then add 3 drops of litmus cream (directly from bottle—no bile salts).  Swirl the contents and leave tube #7 in the ice bath for 30 minutes.

 

19. To tube #8, add 12 drops of buffer, 3 drops of lipase and 3 drops of litmus cream with bile salts (mortar).
20. To tube #9, add 12 drops of buffer and 3 drops of lipase solution. Place tube #9 in a boiling water bath for 5 minutes using a test tube clamp. After 5 minutes, remove the tube.  Then add 3 drops of litmus cream with bile salts (mortar).

 

21. Place tube #10 in the ice bath and add 12 drops of buffer and 3 drops of lipase solution. Let the tube sit for 2 minutes then add 3 drops of litmus cream with bile salts (mortar).  Swirl the contents and leave tube #10 in the ice bath for 30 minutes.

 

22. Swirl all tubes to mix contents.
23. Place all of the tubes (except #7 and #10 which are in the ice bath) in the 37^oC water bath for 30 minutes.

 

I). Testing for the presence of Lipid digestion

1. The basis of this assessment is a pH change detected by the litmus indicator that has been added to the heavy cream. Fats are digested (hydrolyzed) to fatty acids during hydrolysis. The presence of fatty acids lowers the pH of the sample.  If hydrolysis has occurred, the solution will turn from bluish to pink.

 

2. Record color change on data table 3 according to the following:

Blue, clear or yellow                                      (-)

light pink                                                        (+)

medium pink                                                  (++)

bright pink or red                                           (+++)

 

 

 

 

 

 

 

 

 

 

 

 

 

Laboratory Report for Lipid Digestion (Sections E & F Below)

 

Student  Name:____________________________  Date:______________

TA Name:_________________________________

________________________________________________________________

 

1. Discussion Questions (40 points)
2. Describe how the ice bath affects fat digestion?  Did you get the expected results? Explain. (5 pts)
3. Describe how boiling the enzyme affects lipid digestion? Did you get the expected results? (5 pts)
4. Describe how the addition of bile salts affects lipid digestion a) under normal conditions; b) after boiling the enzyme; c) with the ice bath. Discuss/explain your results. (15 pts)
5. Explain how litmus was used in this experiment. How was the detection of lipid digestion achieved in the experimental test tubes? (10 pts)
6. Based on your table, what is the optimum environment for lipase activity (explain)? (5 pts)

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