DEPARTMENT OF PURE AND APPLIED CHEMISTRY

COLLEGE OF ARTS AND SCIENCES

VISAYAS STATE UNIVERSITY
Visca, Baybay City, Leyte

Chem 31a – Biochemistry Laboratory

Name: Jameil D. Magomnang Jr. Date Performed: May 16, 2016

Lab Schedule: M-S (7:00 – 10:00) Date Submitted: May 19, 2016
Group No. 4 Score: __________

Experiment No. 9

CHEMISTRY OF URINE

I. OBJECTIVES:

1. To test urine for pH, specific gravity, and the presence of electrolytes and organic
compounds; and
2. To test urine for the presence of abnormally occurring compounds of proteins, glucose,
and ketone bodies.

II. DATA AND OBSERVATIONS

Table 1. Color, pH, and Specific Gravity

Urine A Urine B

A-1 Color yellow colorless

A-2 pH 6.0 6.0

A-3 Specific Gravity 1.020 1.000

Table 2. Urea

Urine A Urine B

Effect on Litmus Paper red litmus paper turned blue red litmus paper turned blue
Table 3. Uric Acid

Urine A Urine B

Crystals formation of uric acid crystals no uric acid crystals formed

Table 4. Electrolytes

Urine A Urine B

D-1 Cl- (+) (+)

D-2 SO4 2- (+) (-)

D-3 PO4 3- (+ +) (-)

Na+ (+) (+)

D-4
K+ (+) (+)

D-5 Ca2+ (+) (-)

Table 5. Glucose

Urine A Urine B

Benedict’s blue-green blue

Estimate of mg% 0.25% 0.10%

Estimate of mg/dL 250 mg/dL 100 mg/dL

Table 6. Ketone Bodies

Urine A Urine B

Color of Ring presence of brown rings (4 layers) presence of brown rings (3 layers)

Table 7. Protein

Urine A Urine B

Appearance no formation of white precipitate (-) no formation of white precipitate (-)

G. BAR GRAPH OF ELECTROLYTES

350

300
Concenration (mol/L) of Electrolytes in Urine at pH 5.5

Legend:
Na+
250 K+
Ca2+
200 Mg2+
NH4+

150 Cl-
HPO43-

100 SO42-
Organic acids
Urea
50
Creatinine

0
Cations 1 Anions Nonelectrolytes
Ions

Figure 1. Graphical representation showing different ions (electrolytes) and their concentration
(mol/L) in urine at pH 5.5

III. DISCUSSION

The major function of the urinary system is to remove waste products from our bodies and
help regulate the volume and composition of blood by the production and excretion of urine. The
composition of urine can vary greatly and constantly fluctuates with dietary intake (food and
water) and metabolic activity. Simply put, urine is a watery excretion that is composed of waste
products that the body doesn’t really need or hasn’t had the chance to reabsorb. Some might
actually see urine as a sort of expulsion system for the excess water that is in the body, which is
true considering that the majority of the composition of urine is water (around 95 to 96%). But the
main and important consideration is the fact that urine aids in the dumping of chemicals, dead
blood cells and more from the body. So essentially urine is composed of many components with
water serving as the medium that carries the waste out of the body, and it is these that make up the
other 4 to 5%. This laboratory experiment was intended for the observation of the composition of
urine, the interpretation of the amount of each of the individual components of each sample
accordingly, and how the urine samples from each individual differ from each other depending on
certain parameters (illness, etc.). As a guide, urine is considered to have quite a large amount of
urea, followed by the electrolytes like chlorides and sodium. Urine also contains compounds like
uric acid, ketone bodies, proteins and glucose. The quantitative testing of these compounds can
lead to immediate diagnosis depending on circumstances, but this laboratory experiment only goes
so far as qualitative analysis. Each part will be discussed accordingly in the following sections of
the laboratory report.

A. COLOR, pH, AND SPECIFIC GRAVITY

*Color
he color of urine pretty much varies depending on how hydrated the person is, but this can
also more or less be affected by a whole range of different factors. Normally, urine is a colorless
solution that can range from colorless to an amber hue. But there are other factors that can cause
abnormal coloration of the urine, an example being diseases (melanoma producing dark or black
urine) or merely cosmetic coloration from the consumption of certain foodstuffs (like beets
producing pinkish colored urine).
In this part, there were two samples of urine donated by the two group members. For the
first sample (urine A), the color was yellow indicating normal level of hydration. The second
sample (urine B) was observed to be colorless indicating normal level of hydration and excess
amount of water intake. The urine sample showed no turbidity which would have been another
indication of an abnormality.

*pH
The pH of urine usually falls between the ranges of 4.6 to 8, with 7 being the normal pH
level for urine. The pH variations can occur through many ways, but the two of which are
consumption of certain food as well as intake of medications. Consumption of legumes and high
citrus diets have been known to increase urine pH, while diets that are fixed on meat tend to lower
pH levels of the urine.
The pH of the two urine samples as tested with pH paper was found to be 6.0, indicating
normal pH ranges typical of normal urine.
*Specific Gravity

One of the main roles of the kidneys in humans and other mammals is to aid in the clearance
of various water-soluble molecules, including toxins, toxicants, and metabolic waste. The body
excretes some of these waste molecules via urination, and the role of the kidney is to concentrate
the urine, such that waste molecules can be excreted with minimal loss of water and nutrients. The
concentration of the excreted molecules determines the urine's specific gravity. In adult humans,
normal specific gravity values range from 1.003 to 1.030. By itself, specific gravity is basically a
measure of how much more or less dense a particular liquid is compared to water. Using this
parameter in conjunction with urine analysis, it is an important quantitative variable that is
commonly used in the evaluation of kidney function and help to diagnose renal diseases. Increases
in specific gravity (hypersthenuria, i.e. increased concentration of solutes in the urine) may be
associated with dehydration, diarrhea, emesis, excessive sweating, urinary tract/bladder infection,
glucosuria, renal artery stenosis, hepatorenal syndrome, decreased blood flow to the kidney
(especially as a result of heart failure), and excess of antidiuretic hormone caused by Syndrome of
inappropriate antidiuretic hormone. A specific gravity greater than 1.035 is consistent with frank
dehydration. In neonates, normal urine specific gravity is 1.003. Hypovolemic patients usually
have a specific gravity >1.015. Decreased specific gravity (hyposthenuria, i.e. decreased
concentration of solutes in urine) may be associated with renal failure, pyelonephritis, diabetes
insipidus, acute tubular necrosis, interstitial nephritis, and excessive fluid intake (e.g., psychogenic
polydipsia).
The specific gravity of the urine sample was measured using a refractometer which showed
a rating of 3% brix. Based on the data obtained, the specific gravity for urine A is 1.020 which
falls within the normal range of 1.003 and 1.030, and so there are no problems with urine A. The
specific gravity for urine B is 1.000 which does not fall within the normal range. This result
indicates hyposthenuria (decreased concentration of solutes in urine) which may be associated with
some diseases as renal failure, pyelonephritis, diabetes, etc. There are problems with urine B as
observed based on the specific gravity of the sample.
B. UREA

In this investigation, the enzyme urease, breaks down urea to ammonia and carbon dioxide,
was used. Ammonia (NH3) solution has a high pH which can be detected using a litmus paper. The
ammonia produced by the reaction can also be detected by smell. Urea exists in quite a high
concentration in the urine because it is the waste product that is produced from the breakdown of
proteins in the body. So this makes urea one of the components of urine that will always be found
in any sample. Since the laboratory procedure only involved a qualitative method for analysis, so
only the physical characteristic of the testing was observed. When it was heated, a piece of red
litmus paper was placed on the mouth of the test tube containing the urine (both samples urine A
and B), and the conversion from red to blue was an indication of the presence of gaseous ammonia,
and thus the presence of urea as a component of urine. A simple reaction equation for this reaction
is shown below:
urease
H2 O + (NH2)2CO 2NH3 (g) + CO2

Figure 2. Reaction equation of urea reacting with urease producing ammonia

By quantitative standards, the standard amount of urea in urine usually ranges between 12
to 20 grams within a 24 hour period. Abnormal values for these are attributed to diseases, with low
levels being an indication of kidney problems, and higher levels indicating a substantial increase
of protein breakdown in the body.

C. URIC ACID

Uric acid is a chemical produced when your body breaks down foods that contain organic
compounds called purines. Foods and beverages with a high purine content include liver,
anchovies, mackerel, dried beans, beer, and wine. Purines are also created through the natural
process of cell breakdown in the body. Uric acid is basically one of those rare waste products that
are excreted in the urine but in only a very small amount, at least by normal standards. It is actually
considered as one of the penultimate products of the tissue waste in the human body, being the
waste product from the breaking down of purine nucleotides. In simpler terms, uric acid is
produced from the natural breakdown of the body’s cells as well as the food consumed. Most of
the uric acid as is removed from the body in urine, and a small amount of it passes along in the
stool. But certain physical ailments can actually hinder the removal of uric acid from the blood
which can lead to symptoms such as gout, etc. The test performed for this part of the experiment
was simply taking a sample of the urine and adding to it some concentrated hydrochloric acid. The
beaker with the sample and HCl mixture was then left covered with a plastic wrap for a day and
then observed for the formation of any crystals of uric acid. This basically works on the principle
that uric acid is not soluble at extremely acidic pH levels, which in this case would be bought about
by the addition of the concentrated hydrochloric acid.
Most uric acid is dissolved in the blood, filtered through the kidneys, and expelled in the
urine. Sometimes, the body produces too much uric acid or doesn’t filter out enough of it.
Hyperuricemia is the name of the disorder that occurs when you have too much uric acid in your
body. High levels of uric acid are associated with a condition called gout. Gout is a form of arthritis
that causes swelling of the joints, especially in the feet and big toes. Another cause of
hyperuricemia is increased cell death, due to cancer or cancer treatments. This can lead to an
accumulation of uric acid in the body. It’s also possible to have too little uric acid in your blood,
which is a symptom of liver or kidney disease. It’s also a symptom of Fanconi syndrome, a disorder
of the kidney tubules that prevents the absorption of substances such as glucose and uric acid.
These substance are then passed in the urine instead.
Urine A showed formation of some uric acid crystals (little amount) situated at the bottom
of the beaker after 2 days of quantification. Urine B showed no formation of any crystals of uric
acid, indicating a low or close to no amount of uric acid present in the urine. This might be an
indication of kidney problems, meaning that the kidneys aren’t able to get rid of the uric acid well
enough. But this can’t be properly observed with only qualitative testing, since proper parameters
are required for actual diagnosis.

D. ELECTROLYTES

In basic terms, an electrolyte is any substance that contains free ions that behaves as an
electrically conductive medium. All higher life forms cannot exist without electrolytes, and that
includes human beings. But then these only exist in a constant concentration throughout the body
and are constantly replaced by the consumption of food or supplements. Any of the excess
electrolytes are then filtered out by the kidneys and particularly out through the urine. To keep it
simple, electrolytes are important substances because they aid in the regulation of nerve and
muscle function, the hydration of the body, blood pH levels, rebuilding of damaged tissue and
blood pressure. For the experimental procedure, some of the more common electrolytes were tested
for, and they shall be discussed below. Also in reference to the data obtained, the first urine sample
(urine A) used tested positive for the presence of the ions to be discussed, and the second urine
sample (urine B) showed a positive result only on the ions Cl-, Na+, and K+. It showed negative
results on the ions SO42-, PO43-, and Ca2+.

*Chlorides

Considered as one of the important electrolytes in blood by helping in the regulation of

concentration of fluid inside and outside of the cells. The bulk of the chlorides come from the
intake of salt (sodium chloride) and are mostly absorbed by the intestines. The excess leaves the
body through the urine. The test involved the addition of a solution of silver nitrate to the sample
of urine that has been acidified with nitric acid that would ultimately result in the formation of a
white precipitate. The acidification serves the purpose of preventing false positives, because silver
pairs up with carbonates to form a precipitate that is also white in color. Both samples (urine A
and B) produced white precipitates of AgCl in this test. A simple equation of this reaction is shown
below:

Cl- + AgNO3 AgCl (s) + NO3-

Figure 3. Reaction equation of chloride ion with silver nitrate producing silver chloride ppt.

*Sulfates

Sulfates are essential to the body because all cells require inorganic sulfate for normal
function. Sulfates are considered to be among the most important macronutrients in cells, being
the major source of sulfur in many living organisms. Despite the fact that sulfate itself doesn’t
possess a catalytic function or a role in human energy metabolism, there is much evidence to
suggest that sulfates are not metabolically inert molecules and play an essential role in life. The
qualitative test for this in urine is also through the use of the solubility rules, by adding in a solution
of barium chloride to acidified urine. As a result, only urine A showed a positive result evident
with the formation of white precipitates of BaSO4, the urine B showed a negative result. The
equation for this reaction is shown below:

SO42- + BaCl2 BaSO4 (s) + 2Cl-

Fig 4. Reaction equation of sulfate ion reacting with barium chloride producing barium sulfate
*Phosphates

Phosphates are important because they aid in building and repairing bones and teeth, aid in
muscle contraction and helps nerve function. Much like the other electrolytes, the phosphates are
regulated by the kidneys and any excess amounts are excreted through the urine. This was tested
in the experimental procedure through reaction of the acidified urine sample with ammonium
molybdate, which produced a yellow precipitate indicating the presence of phosphates. The
ammonium molybdate ((NH4)2MoO4) forms a precipitate of ammonium phosphomolybdate
((NH4)3PO4.12MoO4) which is a bright yellow compound that is extremely insoluble even in dilute
nitric acid. As a result, urine A produced many yellow precipitates indicating a positive result.
Urine B showed a negative result in this test. Negative result indicates the occurrence of either
hypomagnesemia, hypokalemia, severe burns, traumatic injuries, chronic alcoholism, kidney
disease, hypothyroidism, malnutrition, or prolonged diuretic use.

*Sodium and Potassium

These two cations are basically considered to be the power generators inside the cells of
the body, and they are invaluable in the process of neural transmission. The presence of the two
electrolytes in urine can be tested for qualitatively through the use of a simple flame test. A flame
test wire was dipped into some hydrochloric acid and heated to red hot in the flame of a Bunsen
burner. This was done to prevent any form of false positives. The wire was then placed into the
urine solution and then heated. The formation of a yellow flame indicated the presence of sodium
and the formation of a discernable red flame as seen through a cobalt glass square indicated the
presence of potassium in the urine sample. As a result, both urine samples (urine A and B) showed
positive results for this test.

*Calcium

This particular electrolyte plays a vital role in signal transduction pathways (activation of
receptors by an external signaling molecule that can alter the cell’s metabolism, etc.). Calcium also
plays an important role in the contraction of all muscle cell types and in some other important
processes such as fertilization. With regard to the experimental procedure, this electrolyte was test
for on the basis of the solubility rules, where a urine sample was acidified with acetic acid and then
reacted with ammonium oxalate. The formation of a white precipitate of calcium oxalate indicated
a positive result for the urine sample. As a result, only urine A showed a positive result for this
test evident with the formation of cloudy, white precipitate of calcium oxalate; urine B showed a
negative result. Calcium deficiency can cause kidney failure thyroid disorders, vitamin D
deficiency, and medications like heparin. Shown below is a simple equation for the reaction:

Ca2+ + (NH4)2C2O4 CaC2O4 (s) + 2NH4+

Figure 5. Reaction equation of calcium ion reacting with ammonium oxalate producing calcium
oxalate precipitate

E. GLUCOSE

The normal amount of glucose in urine is 0 to 0.8 mmol/L (millimoles per liter). A higher
measurement could be a sign of a health problem. Diabetes is the most common cause of elevated
glucose levels. Glucose is a compound that usually not found in urine because of the normal
capability of the kidneys to reclaim all of the filtered glucose back into the blood stream. The
presence of glucose in the urine is basically condition that is known as glucosoria, which is a
condition caused by elevated blood glucose levels that are brought about by untreated diabetes
milletus. A simple and functional test for this would be the Benedict’s test for reducing sugars.
Based on the laboratory results, the first urine sample (urine A) formed a blue-green color when
mixed with the Benedict’s reagent. Based on the table of values, blue-green represents 0.25% of
milligrams of glucose present or 250 mg/dL. The second urine sample (urine B) used didn’t change
from the original blue color it had when mixed with the Benedict’s reagent, which indicates a low
level or close to none depending of the amount of cupric oxide formed as necessary. Based on the
table of values, blue represents 0.10% of milligrams of glucose present or 100 mg/dL. A simple
illustration of this reaction (taking into consideration a urine sample that has a high amount of
glucose) is shown below:
F. KETONE BODIES

Ketone bodies are acids made when your body begins using fat instead of carbohydrates
for energy. When there is not enough insulin to get sugar from the blood and into the cells, the
body turns to fat for energy. When fat is broken down, ketone bodies are made and can accumulate
in the body. High levels of ketones are toxic to the body. The condition is called ketoacidosis.
Ketones are most likely to show up when there is not enough insulin in the body. This can happen
if people who have type 1 diabetes don’t take insulin or don’t take enough to meet higher demands,
such as during illness or stress, or when a pump gets clogged or unattached. It can also happen in
people with type 2 diabetes who are insulin-deficient if they get sick. In normal urine, ketone
bodies will not be present. In the experimental procedure, the first urine sample (urine A) tested
positive for ketone bodies, which could be an indication of a medical condition known as
ketonuria; urine B showed a negative result for this test. The test for this particular compound in
the urine is known as the Rothera’s Test which is a qualitative test to test for ketone bodies. The
urine sample was saturated with ammonium sulfate in order to concentrate the ketone bodies to
the center of the solution and also has an added function of the ammonium ions being able to
precipitate any proteins that might be present (false positives). Nitroprusside was then added to
this mixture, followed by the gradual addition of ammonium hydroxide or ammonia solution. The
slight formation of a ring with a formation of 4 layers for urine A and 3 layers for urine B was
observed at the junction between the urine and ammonia/ammonium hydroxide solution. The ring
is a product of the complex between the ketone and the nitroprusside in the presence of ammonia.

G. PROTEINS

In terms of the presence of proteins in urine, healthy individuals usually don’t have any
protein in their urine, which is facilitated through the regulation of the proteins by the kidneys,
which basically ensure that they remain in the blood stream. A small amount of proteins is usually
not much for concern, but high amounts of protein in urine are indications of kidney failure,
diabetes or urinary tract infection. For the laboratory experiment, the upper portion of the urine
sample was heated, and with the initial formation of turbidity 5 drops of acetic acid was added
(these are false positives). The heating of the same upper portion a second time showed no
evolution of a faint turbidity which indicated a negative result. Both urine samples (urine A and
B) showed negative results in this test evident with no formation of white precipitate. The result
indicates that the urine is healthy due to the fact that the presence of protein was not observed in
this test.

IV. CONCLUSION

After the conduct of this experiment, it is concluded that the normal color of urine ranges
from colorless to yellow; pH of urine is 6.0; and its specific gravity ranges from 1.003 to 1.030.
The presence of electrolytes and organic compounds in urine is normal, unless they are in high or
low amount which causes abnormalities in the system of the body. It is also concluded that the
presence of high amounts of glucose and ketone bodies detects the occurrence of some
abnormalities in the systems of the body and may lead to the formation of some diseases such as
diabetes mellitus and glucosoria (for glucose), and ketonuria (for ketone bodies). In terms of the
presence of proteins in urine, healthy individuals usually don’t have any protein in their urine,
which is facilitated through the regulation of the proteins by the kidneys, which basically ensure
that they remain in the blood stream. Based on the results obtained, it is concluded that the two
urine samples are healthy (in terms of protein content in urine). Urine is a fluid that contains waste
products from the human body whose components are pretty diverse. Over the years, the use of
urine in making diagnosis related to the condition of the body has been paramount and has
developed even more throughout medical history. This experiment dealt with the observation of
the different components that are to be found in urine, but only in the qualitative perspective.

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