Investigation of the Chemical Components of Urine

(Urinalysis)

Adapted from:1) Urinalaysis, Greg Henkelman, Edmonton Public School Board

To pee or not to pee!To Pee or Not to Pee…what Shakespeare really wanted to write.

To pee or not to pee, that is the question.Whether tis nobler in the mind to suffer

The slings and arrows of a kidney stone passageOr to take arms against the stone, and by sound waves, break it into smaller pieces.

And by doing so, pass them. Later, we’ll decide.No more on that; and now we look at urine.

The heart-beat and the million nephron unitsThat kidneys require, tis a working mechanism

Devoutly to be wish’d. To diffuse, passivelyTo transport, actively. Ay, there’s the rub.For after filtration through the glomerulus

When we have entered into the nephron’s coils,We must give pause and consider what is to pass in and out,

That makes an easiness of so long a life.For who would bear the toxicity of urea filled blood,

Th’ammonia’s wrong, the salty presence,Without pangs, the glucose, urea, and salts,

The water, fatty acids and glycerols,Enter into Bowman’s capsule,

There to continue into the proximal convolutions.Osmosis pulls water in, to the hypertonic blood.

To grunt and sweat under a weary life,Is one way we use ATP, but

It is also used to transport glucose and amino acidsInto the undiscover’d country of the bloody network, from whose bourn

No traveller returns. And protein, pinocytosed,Gives us the material to bear those ills we have.But what of the water remaining in the nephron?

The Loop of Henle is the answer (at least in mammals and birds).The concentration of the medulla being

Greater than the cortex, water leaves the nephron,Passively, just like salt enters.

Then, Cl- is pumped out of the ascending loopWater, K+, and Na+ follow.

With this now done, the urine’s current turns awayTossed in the cortex distal tubule.

Where aldosterone, from adrenals, works its magic,Encouraging salty readmission of Na+,

By which water soon follows.And now, into the salty environs

Of the medulla again.The collecting duct, controlled in part by ADH,

And controlling the pH, is made to open its armsReleasing water into the blood.

But soft you now, the fair excretion will be made,Upon completion of this coffee in my grasp.

-G. Henkelman (an adaptation on a slightly more famous soliloquoy)Edmonton Public School Board

BACKGROUND

Homeostasis is the maintenance of a relatively stable internal environment. Just as sweat

glands in your skin help to regulate body temperature through the excretion of water, and

your lungs regulate oxygen/carbon dioxide concentrations through the elimination of

carbon dioxide, your kidneys contribute to the maintenance of internal homeostasis by

regulating the volume, pH and composition of blood, while removing water and metabolic

wastes. Urine is a waste or by-product of the kidneys' activities. The volume and

composition of normal urine provides a good indicator of a person's overall health.

In humans, urine is made up primarily of water and dissolved salts and organic materials.

The concentration of urine's constituents varies with a person's health, diet and degree of

activity.

Urinalysis is an examination of urine's physical and chemical characteristics. By testing the

chemical composition of urine, doctors can learn much about the general health of an

individual.

This type of examination is necessary to correctly diagnose potentially harmful chemicals,

chemical imbalances and pathogens as they appear in the excretory system. Results from a

standard urinalysis may be used to detect, among other

things, drug abuse, pregnancy and kidney damage due

to the presence of toxins, trauma or infection. Urinary

tract infections, kidney malfunction, and liver disease

are just some of the medical problems that can be

diagnosed through urinalysis. Additionally, urinalysis is

often performed in tandem with blood tests; results

obtained in these tests often correlate with one another

and help provide medical professionals with additional

information.

Normal urine is a transparent solution with a light yellow to amber color. The color of urine

is strongly influenced by concentration, diet and hormones. The detection of physical

abnormalities such as red blood cells or uncharacteristic odors in a

urine sample often provides medical professionals with a starting

point for further investigation and assessment. For example, a

urine sample with a "sweetish" odor suggests the presence of

unusually high amounts of ketone bodies and may provide a

preliminary indication of diabetes. The suffix "-uria" indicates

excess urinary excretion. Acetonuria (ketosis) is the excretion of

unusually high amounts of ketone bodies. While diabetes mellitus

may be suspected when acetonuria is detected, starvation or

insufficient intake of carbohydrates may also increase ketone excretion, as will genetically

or chemically acquired metabolic abnormalities. Other physical abnormalities, such as the

presence of bacteria and other microbes, also give rise to the need for further investigation.

Bacteria in urine may be observed through culturing and staining with Eosin Methylene

Blue (EMB). Samples may be spun in a centrifuge. Examination of these residues may

indicate the presence of mineral crystals, yeast, bacteria, red or white blood cells or casts.

Yeast in urine indicates a yeast infection of the urinary tract.

In addition to a physical examination, most urine samples undergo

several chemical tests to determine composition and solute

concentrations. Many chemical tests involve the use of test strips

specifically designed to evaluate pH, concentrations of glucose,

ketones, bilirubin, urobilinogen, plasma proteins and hemoglobin.

Chemically, we can detect the presence of albumin (protein),

glucose, yeasts and evaluate pH using biuret solutions, Benedict's

solution, hydrogen peroxide and pH strips, respectively. The pH of

normal urine ranges from 4.8 to 7.5; the average pH is about 6.6.

Consistently acidic urine (low pH) may indicate conditions such as

metabolic or respiratory acidosis, uncontrolled diabetes,

starvation, and dehydration. Consistently alkaline urine (high pH)

may indicate conditions such as metabolic or respiratory alkalosis,

urinary tract infections, or renal failure. Albumin (protein) in urine indicates possible

kidney disease. Glucose in urine may be caused by temporary high consumption of sugar or

simple starches, or by diabetes mellitus. (Blood glucose tests would be performed to

confirm a diagnosis of diabetes.)

In this laboratory exercise, you will use a variety of chemical and physical techniques,

including your senses, to identify both normal urine and urine suspected to have abnormal

constituents.

OBJECTIVE: BECOMING A MEDICAL DETECTIVE

Your task in this laboratory is to become a "sleuth." Sleuths are detectives, trackers or

"bloodhounds" who seek to find a criminal, a "bad guy" or a missing object. Think of

yourself as Sherlock Holmes on an important case. In your lab, you will "search" synthetic

urine samples for physical and chemical "criminals" (abnormal chemical concentrations and

microbes). To be a good detective, you will need to use your powers of observation and

deduction, as well as your senses (sight, smell) to identify unknown chemical contaminants

in various urine samples.

Throughout these labs it is important that you use proper procedures for working with

chemicals and lab ware. These include wearing a laboratory apron and safety goggles and

handling all glassware carefully. Also, use extreme care when working with heated

equipment or materials to avoid burns. Always use extreme caution when working with

laboratory chemicals, as they may irritate the skin or cause staining of the skin or clothing.

Never touch or taste any chemical unless instructed to do so. Observe proper laboratory

procedures when using electrical appliances.

PART I: ESTABLISHING POSITIVE CONTROLS

Before you can analyze clues to track down "criminals" in urine, you need to know how the

presence of these criminal elements affects normal urine. In this part of the lab, you will

establish positive controls.

Benedict's Test for Glucose

1. Place two clean test tubes in a test tube rack. With a glass-marking pencil, label one

test tube "G" for glucose. Label the other test tube "C" for control.

2. Fill a 200-mL beaker approximately half full of water and heat to boiling then TURN

OFF THE HEAT. Caution: Use extreme care when using boiling hot water to avoid

splashing it on yourself or your lab partner.

3. Add 2 mL of urine with glucose and add 10 drops of Benedict's solution to the test

tube marked “C”. Caution: Avoid direct contact with Benedict's solution.

4. Add 2 mL of the urine without glucose to the test tube labeled "G." Add 10 drops of

benedict’s solution. Carefully swirl each tube to mix.

5. Carefully place both test tubes upright in the hot water bath (the beaker of heated

water). Take care that the contents of the test tube do not spill into the beaker, and

that the water from the hot water bath does not overflow into the test tubes.

6. After 2 minutes, carefully remove both test tubes from the hot water bath with a test

tube holder. Place the test tubes in the test tube rack. Caution: Be careful when

working with heated equipment or materials to avoid burns. Note the color of the

sample and the presence or absence of a precipitate in each of the test tubes and

record your observations in the appropriate spaces in Data Table I.

Test for Albumin (Protein)

1. Place two clean test tubes in a test tube rack. With a glass-marking pencil, label one

test tube "A" for albumin. Label the other test tube "C" for control.

2. Add 2 mL urine with albumin to test tube "A." Add 2 mL normal urine to test tube

"C."

3. Add 10 drops biuret solution to both test tube "A" and test tube "C." Swirl or agitate

each tube to mix.

4. Note the appearance of the contents of each tube. A positive test for protein (such as

albumin) is the development of a deep violet color. Record your observations for

each test tube in the appropriate spaces of Data Table I.

Test for Yeast

1. Place two clean test tubes in a test tube rack. With a glass-marking pencil, label one

test tube 3 "Y" for yeast. Label the other test tube "C" for control.

2. Add 2 mL urine with yeast to test tube "Y." Add 2 mL normal urine to test tube "C."

3. Carefully add 20 drops hydrogen peroxide solution to each test tube. The formation

of bubbles is a positive test result for the presence of yeast. Observe the appearance

of each sample, and note whether bubbles formed or not. Record your results in the

appropriate spaces of Data Table I.

pH Test

1. Place two clean test tubes in a test tube rack. With a glass-marking pencil, label one

test tube "c" for normal pH. Label the other test tube "p" for test solution.

2. Add 2 mL of the urine with unknown pH to the test tube labeled "p". Add 2 mL of

normal urine to the test tube labeled "C".

3. Dip one pH test strip into test tube "p." Dip a second pH test strip into test tube "C."

Wait 30 seconds and compare the colors of the test strips with a pH chart provided.

Record your pH readings in the appropriate spaces of Data Table I.

Ketone Test

1. Place two clean test tubes in a test tube rack. With a glass-marking pencil, label one

test tube "c" for normal urine. Label the other test tube "K" for the test solution.

2. Add 2 mL of the urine with acetone to the test tube labeled "K." Add 2 mL of normal

urine to the test tube labeled "C". Using your hand, make a sweeping motion over

the top of each test tube to direct the odor of each sample toward your nose. Note

the odor of the urine sample in each test tube, and record your observations in the

appropriate spaces of Data Table I.

PART II: TESTING FOR UNKNOWNS AKA. PLAYING HOUSE.

Using your data (the background information) from Part I, you will now work to identify

unknown chemical contaminants in various urine samples.

1. Obtain 10 mL of each of the unknown samples in a clean labeled beaker. (ie. label

the beakers from 1-5) Make sure that each of the solution has been stirred

A. Benedicts Test for Glucose

1. Place 2 mL of each of the unknown samples in each of five test tubes labeled from 1

to 5.

2. Add 20 drops of Benedict's solution to each of the test tubes." Caution: Avoid direct

contact with Benedict's solution. Carefully swirl or agitate the tube to mix.

3. Carefully place test tube the labelled test tubes in a hot water bath for 2 minutes.

Take care that the contents of the test tubes do not spill into the beaker, and that the

water from the hot water bath does not overflow into the test tubes.

4. Carefully remove the test tubes 1-5 from the boiling water bath and observe the

contents of the tube. Using the positive controls you established for the presence of

glucose (refer to Data Table I), determine whether this "criminal" is contaminating

sample any of your samples from 1-5. Record your results for the presence of

glucose in the appropriate spot, ie Sample #1 -(+) for a positive result and (-) for a

negative result - on Data Table II.

5. Clean up the test tubes.

B. Biuret’s test for protein

1. Again add 2 mL of each unknown into each test tube (labeled from 1-5).

2. Into each test tube, carefully add 10 drops biuret solution. Carefully agitate the tube

to mix its contents.

3. Using the positive controls you established for the presence of protein (refer to Data

Table I),

4. Determine whether this "criminal" is contaminating any of your samples. Record

your results for the presence of protein in each sample; (+) for a positive result and

(-) for a negative result –in the appropriate space in Data Table II.

5. Clean up the test tubes.

C. Test for yeast.

1. Again add 2 mL of each unknown into each test tube (labeled from 1-5).

2. Into each test tube carefully add 20 drops hydrogen peroxide solution. Using the

positive controls you established for the presence of yeast (refer to Data Table I),

determine whether this "criminal" is contaminating any of your samples. Record

your results for the presence of yeast in each sample: (+) for a positive result and (-)

for a negative result -in the appropriate space in Data Table II.

3. Clean up the test tubes.

D. pH test

1. Again add 2 mL of each unknown into each test tube (labeled from 1-5).

2. Dip one pH test strip into each sample from 1-5. Wait 30 seconds. Using the positive

controls you established for a sample of normal acidity (refer to Data Table I),

determine whether a "criminal" acid is contaminating any of your samples. Record

your pH reading for each sample in the appropriate space in Data Table II.

Ketone test

1. Again add 2 mL of each unknown into each test tube (labeled from 1-5).

2. Using your hand, make a sweeping motion over the top of test tube "1 e," to direct

the odor of the sample toward your nose. Using the positive controls you

established for the presence of ketones (refer to Data Table I), determine whether

this "criminal" is contaminating each of your samples. Record your results for the

presence of ketones in each sample with a (+) for a positive result and H for a

negative result -in the appropriate space in Data Table II.

3. Clean up the test tubes.

Conclusion and Evaluation

In your write up:

States a conclusion on the content of the samples with justification based on a

reasonable interpretation of the data.

Evaluates weaknesses and limitations of the laboratory exercise.

Suggests realistic improvements in respect of identified weaknesses and limitations.