Experiment

Metabolism
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INTRODUCTION

Metabolism is defined as the sum of all biological processes occurring in a living

system. Reactions involved during metabolism are sub-classified into anabolism

and catabolism.

Digestion is the breaking-down process that takes place in various parts of our

body: in the mouth, the stomach, and the intestines. Starch, fats, and proteins are

broken down first into their building constituents. All digestive processes are

induced by enzymes which are contained in the digestive juices – the saliva,

gastric juice, and pancreatic juice. In our mouth, the food gets mechanically

crushed and mixed with saliva. Saliva contains the enzyme ptyalin, an amylase.

This enzyme cleaves starch into sugar.

After the food has been chewed in the mouth, it goes through the stomach.

Digestion of proteins begins in the stomach. The gastric juice contains the enzyme

pepsin and diluted hydrochloric acid which activates pepsinogen, the precursor of

the enzyme pepsin. The proteins are cleaved by the enzyme pepsin into peptides

which are later further digested in the intestines.

In the intestines, the already pre-digested food is further digested and finally

the nutrients are absorbed through the intestinal wall. The enzymes, involved in

the digestion of the food pulp in the intestine, are produced by the pancreas and

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released as pancreatic juice into the duodenum. The pancreatic juice contains

lipases (fat splitting enzymes), amylases (starch splitting enzymes), as well as

proteases (protein splitting enzymes).

Pyruvate, which is the main product during the breakdown of glucose in

glycolysis, under aerobic conditions, is further oxidized in the citric acid cycle upon

the formation of acetyl Co-A from oxidative decarboxylation facilitated by pyruvate

dehydrogenase enzyme complex. However, during anaerobic conditions, pyruvate

undergoes fermentation depending on the type of organism involved. In most

unicellular organisms, pyruvate undergoes alcoholic fermentation, producing

ethanol from the action of alcohol dehydrogenase enzyme.

OBJECTIVES

At the end of the experiment, the student should be able to:

1. Demonstrate knowledge about the basic concept of metabolism; and

2. Observe different enzyme-catalyzed reactions in digestion.

MATERIALS/APPARATUS

Erlenmeyer flask, weighing scale, graduated cylinder, beaker, test tubes,

water bath, hot plate, stirring rod, dropper

WASTE DISPOSAL

 Flush acid solutions with plenty of water.

 All solid materials are to be thrown into the trash can.

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EXPERIMENTAL PROCEDURE

A. Preparation and extraction of starch

1. Peel and grate a medium-sized potato/sweet potato.

2. Filter through a cheesecloth and collect the filtrate (prepared starch)

using a beaker.

3. Put 5 drops of the prepared starch solution into a test tube and add 2

drops of Lugol’s Iodine.

4. Observe the change in color.

B.1 Salivary Hydrolysis

1. Collect 5mL of saliva and add 3 mL of the prepared starch solution.

2. Place a drop of iodine and stir from time to time until the violet color

disappears.

3. Test with 5 mL of Benedict’s reagent and warm in a water bath for 5

minutes.

4. Record your observation

B.2 Acid Hydrolysis

1. Place 10mL of the prepared starch solution into a beaker.

2. Dilute with 40mL of distilled water, and add 1mL of concentrated HCl.

3. Boil the mixture continually until it does not form a purple complex when

tested with Lugol’s Iodine.

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 4. Add 1-2 drops of Phenolphthalein. Carefully add NaOH until it becomes

neutral as indicated by a pale pink endpoint.

5. Test 2mL of the hydrolyzed starch with 5 mL of Benedict’s reagent and

warm in a water bath for 5 minutes. Record your observations.

B.3 Alcoholic Fermentation of Hydrolyzed Starch

1. Put 20mL of the prepared starch solution into an Erlenmeyer flask, and

add 2g of brewer’s yeast.

2. Cover and seal with a balloon and incubate for 24 hrs at room

temperature.

3. Observe what happens to the balloon after 24 hours of incubation.

C. Digestion in the Stomach

1. Fill four test tubes as follows:

Test tube A: 10 mL water

Test tube B: 9 mL of 1% pepsin solution and 1 mL of water

Test tube C: 9 mL of water and 1 mL of 5% hydrochloric acid

Test tube D: 9 mL of 1% pepsin solution and 1 mL of 5% hydrochloric acid

2. Put into each test tube a piece of boiled fish/egg white about the size of a pebble.

3. Warm the test tubes in a water bath maintained at 35-40°C for 1 hour.

4. Afterwards, leave the test tubes at room temperature for 24 hours.

5. Check the texture of the samples using a glass stirring rod and record.

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6. Test the digested materials with Biuret reagent by getting 3 mL of the material

in the lower portion of the tube, and then add 2 mL of 40% NaOH and 2 drops of

1% CuSO4.

7. Compare the color of the solutions formed.

D. Digestion in the Intestines

D.1. Starch Digestion using Pancreatic Amylase

1. Put 3 mL of the prepared starch solution into two test tubes.

2. Place a drop of Lugol’s Iodine into each tube. Shake and note the color

of the solutions.

3. Into one test tube, add 5 mL of water and to the other, 5 mL of 1 %

pancreatin suspension.

4. Cover the tubes with parafilm and mix the contents.

5. Closely observe the changes in color in the next minutes and record

your observations.

6. Test the solutions with 5 mL of Benedict’s reagent and warm in a water

bath for 5 minutes.

7. Record your observation

D.2. Protein Digestion using Trypsin

1. Fill two test tubes as follows:

Test Tube A: 5 mL of 0.5% sodium carbonate solution and 5 mL of 1%

pancreatin dispersion.

Test Tube B: 5 mL of 0.5% sodium carbonate solution and 5 mL of water.

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2. Then put into each test tube a piece of boiled fish/egg white about the

size of a pebble.

3. Warm the test tubes in a water bath maintained at 50°C for 1 hour.

4. Afterwards, leave the test tubes at room temperature for 24 hours.

5. Check the texture of the samples using a glass stirring rod and record.

6. Test the digested materials with Biuret reagent by getting 3 mL of the

material from the lower portion of the tube, and add 2 mL of 40% NaOH and

2 drops of 1% CuSO4.

7. Compare the color of the solutions formed.

D.3. Fat Digestion using Pancreatic Lipase

1. Fill two test tubes as follows:

Test Tube A: 5 mL of cream, 5 mL of 1% pancreatin dispersion and 2 mL of

bromthymol blue.

Test Tube B: 5 mL of cream solution, 5 mL of water and 2 mL of bromthymol

blue.

2. Mix thoroughly and warm at 50oC.

3. Observe for changes in the color of the solution and odor.

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 Report: Name:

Experiment

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Section: Group:
Metabolism Date:

Instructor/s:

DATA AND RESULTS

A. Hydrolysis of Starch

Prepared Starch
Iodine Test

Hydrolyzed with saliva

Benedict’s Test Hydrolyzed with acid

Fermentation

B. Digestion in the Stomach

Observation

Test Tube A

Test Tube B

Test Tube C

Test Tube D

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C. Digestion in the Intestines

Starch Digestion using Observation

Test Tube A

Test Tube B

Protein Digestion using Observation

Test Tube A

Test Tube B

Fat Digestion using Observation

Test Tube A

Test Tube B

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QUESTIONS

1. How does the digestive process for carbohydrates, lipids, and proteins

occur?

2. What are the different fates of pyruvate? How does it differ in prokaryotes

and in eukaryotes?

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 Experiment

11 Urine
INTRODUCTION

Urine is a waste product from the filtration of blood in the kidney. Majority of the

chemical constituents of the urine is water while urea is considered as the major

organic constituent. Other forms of substances such as inorganic salts and organic

compounds are also present. The relative amount or concentration of substances

in the urine may vary in terms of threshold. Since the urine reflects the efficacy of

the kidney to filter the blood, other constituents that may be found in the urine may

reflect important diseases. This may include glucose, proteins, ketones, bile, and

bile acids. Thus, the analysis of urine has been a cornerstone in the diagnosis of

normal renal function.

OBJECTIVES

At the end of the experiment, the students should be able to:

1. Identify important physical properties of urine; and

2. Determine the presence of some normal and abnormal organic and

inorganic constituents of urine.

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MATERIALS/APPARATUS

Test Tube, test tube rack, test tube holder, dropper, stirring rod, water bath,

graduated cylinder, hot plate, beaker

WASTE DISPOSAL

 Flush acid solutions with plenty of water.

 All solid materials are to be thrown into the trash can.

EXPERIMENTAL PROCEDURE

A. Collection of Urine Sample

1. In a clean beaker, collect freshly voided midstream clean catch urine

sample.

2. The midstream clean catch is the middle portion urine sample during

micturation.

3. Take extra precautions in handling the urine sample. ALWAYS remember

to treat all biological samples as potentially infectious.

B. Physical Properties

1. Observe for the color, transparency, and odor of the urine sample.

2. Test the pH of the urine sample using a pH paper.

3. With the use of a urine strip, test for the specific gravity of the urine

sample.

4. Record your observation.

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C. Qualitative Analysis of Normal Urine Constituents

C.1 Test for Chlorides

1. Place a blue litmus paper into 3 mL of the urine sample in a test tube.

2. Acidify the urine by adding 2N Nitric acid drop wise.

3. Add 4-5 drops of Silver nitrate solution.

4. Note any reaction and record your observation.

C.2 Test for Sulfates

1. Place a blue litmus paper into 3 mL of the urine sample in a test tube.

2. Acidify the urine by adding 5N Hydrochloric acid drop wise.

3. Add 15% of Barium Chloride solution drop by drop.

4. Note any reaction and record your observation.

C.3 Test for Phosphates

1. Place a red litmus paper into 5mL of the urine sample in a test tube.

2. Make the urine alkaline with 10% Ammonium Hydroxide.

3. Note any reaction and record your observation.

C.4 Test for Urea

1. Heat 5 mL of the urine sample to concentrate its components into a

test tube.

2. Stop heating when the volume is approximately reduced to 2-3 mL.

3. Cool and add 5mL of 10% Sodium Hydroxide.

4. Add 6-10 drops of 1% Cupric Sulfate solution.

5. Note any reaction and record your observation.

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 C.5 Test for Creatinine

1. Place 5mL of the urine sample into a test tube.

2. Add 1mL of saturated picric acid solution and 1 mL of 2N Sodium

Hydroxide.

3. Note any reaction and record your observation.

D. Qualitative Analysis of Abnormal Urine Constituents

NOTE: In all of the succeeding tests, prepare a positive control urine sample

for comparison.

D.1 Test for Albumin

1. Place 3 mL of urine sample into a test tube and heat at the mid portion

until boiling.

2. Observe for the appearance of turbidity.

3. Add 3-4 drops of 5% acetic acid, if the precipitate disappears, it is due

to phosphates and carbonates.

4. If the precipitate remains, urine sample is positive for albumin.

5. Repeat the entire procedure using a positive control urine sample.

6. Compare, record, and interpret the results observed.

D.2 Test for Glucose

1. Place 2mL of your urine sample into a test tube and 2mL of the

positive control urine sample into two separate test tubes.

2. Add 3mL of Benedict’s reagent into each tube.

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3. Warm the test tubes over a water bath for 5-10 minutes.

4. Compare, record, and interpret the results observed.

D.3 Test for Ketones

1. Place 2mL of your urine sample into a test tube and 2mL of the

positive control urine sample into two separate test tubes.

2. Add 2 mL of 5% Ammonium sulfate and 3 drops of 5% Sodium

Nitroprusside into both tubes.

3. Using a red litmus paper, turn the solution into an alkaline one by

adding 1-2mL of Ammonium Hydroxide.

4. Compare, record, and interpret the results observed.

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 Report: Name:

Experiment

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Section: Group:
Urine Date:

Instructor/s:

DATA AND RESULTS

A. Physical Properties

Property Observation

Color

Transparency

Odor

pH

Specific gravity

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B. Qualitative Analysis of Normal Urine Constituents

Analyte Result Interpretation

Chlorides

Sulfates

Phosphates

Urea

Creatinine

C. Qualitative Analysis of Abnormal Urine Constituents

Analyte Positive Control Urine Sample

Albumin

Glucose

Ketones

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QUESTIONS

1. What substances are responsible for normal urine color?

2. How is the specific gravity of the urine affected by its contents?

3. What conditions are closely associated with the presence of glucose in the

urine?

4. What does the presence of albumin in the urine indicate?

5. How relevant is monitoring for ketone bodies in Diabetes mellitus?

What does the presence of these substances in the urine indicate?

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