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Evangelista, Mary Cyrille; Garcia, Kristine Micah Luisa; Go-oco, Racquel; Guillermo, Linette*
College of Science
University of Santo Tomas
Espaa Blvd., Manila
Urine is a liquid waste product of the body secreted by the kidneys by a process of filtration from blood called urination and
excreted through the urethra. Urinalysis is an array of tests performed on urine and one of the most common methods of
medical diagnosis. The objectives of this experiment are to subject the urine sample acquired to several tests and to
qualitatively examine the presence of some normal organic constituents and pathologic organic constituents. Initial
examination includes the notation of the collection time, color, turbidity, acidity and pH of the urine sample. For the
qualitative exam of normal organic constituents, the test for urea, uric acid, indican and creatinine were conducted. In these
tests, the patients results were all normal. For the qualitative examination for pathologic organic constituents, Gunningss
test, Benedicts test, Extons test, Smiths test and Occult blood test were performed. The result obtained for the urine sample
was that the patient was healthy except for the fact that the patient might have diabetes.

Urine is a transparent solution that can range from colorless to amber but is usually pale yellow. Urine is
an aqueous solution of metabolic wastes such as urea, dissolved salts, and organic compounds produced
by the kidneys. It plays a vital role in maintaining homeostasis. The production of urine is called diuresis
(Maxted et al., 2005).

Urine ranges from pale yellow to amber because of the pigment urochrome. The color indicates the
concentration of the urine and varies with specific gravity. Dilute urine is straw colored, while
concentrated urine is deep amber (Pagana & Pagana, 2002).

The aromatic odor of fresh, normal urine is caused by the presence of volatile acids. Analysis of the pH
of a freshly voided urine specimen indicates the acid base balance. The urine reflects the work of the
kidneys to maintain normal pH homeostasis (Pagana & Pagana, 2002). The kidneys maintain normal

acid base balance primarily through reabsorption of sodium and tubular secretion of hydrogen and
ammonium ions (Dunning & Fischbach, 2004).

Urinalysis is part of routine diagnostic and screening evaluations performed on urine that provide a
general overview of a persons health (Tuchman, 2005). Urinalysis is used as a diagnostic tool because it
can help detect substances or cellular material in the urine associated with different metabolic and
kidney disorders. It is routinely done in all patients admitted to the hospital, pregnant women and
presurgical patients. It is done diagnostically in patients with abdominal or back pain, dysuria,
hematuria, or urinary frequency. It is part of routine monitoring in patients with chronic renal disease
and some metabolic diseases (Pagana & Pagana, 2002).

Most urine tests are performed for one of the following reasons: to diagnose renal or urinary tract
disease; to monitor renal or urinary tract disease; to detect metabolic or systematic diseases not directly
related to the kidneys (Pagana & Pagana, 2002).

The process of urinalysis determines the following properties of urine: color, odor, turbidity, specific
gravity, pH, glucose, ketones, blood, protein, bilirubin, urobilinogen, nitrite, leukocyte estaerase, and
other abnormal constituents revealed by microscopic examination of the urine sediment (Dunning &
Fischbach, 2004).

This experiment aims to subject the urine sample acquired to several tests and to qualitatively examine
the presence of some normal organic constituents and pathologic organic constituents.

For the initial examination of urine, the color, turbidity, acidity and the time when the urine was
collected was noted. For the qualitative examination of normal organic constituents, various tests were
performed. For the test for urea, 0.5mL of 70% NaOH and 4 drops of bromine water was added to 1mL
urine sample. Then, the evolution of N2 gas was observed. For the test for uric acid, 1mL of urine sample
was added with 5mL of 20% Na2CO3. Then, 5 drops of phosphotungstic acid reagent was also added
then mixed. The formation of a blue color was observed. For the indican test, 5mL of Obemayers
reagent was added to 5mL urine sample and mixed. Then, 3mL of chloroform was added to it before
shaking and allowed to settle. The formation of a blue color in the lower chloroform layer was observed.
For the test for creatinine, 1mL alkaline picrate solution was added to 2mL urine sample. The formation
of an orange colored solution was noted.

For the qualitative examination for pathologic organic constituents, various tests were also performed.
For Gunnings test, 5mL of urine sample was basified with 5 drops of concentrated ammonium
hydroxide using a red litmus paper. Then, Lugols solution was added to the basic urine, enough to
produce a black cloud which does not disappear immediately. It was let stood for 5 minutes. Similar
procedure was done for the positive control. The results obtained were compared. For Benedicts test,
5mL Benedicts reagent was added to 8 10 drops of urine sample in a test tube then mixed. It was then
heated in a boiling water bath for 2 3 minutes then let stood and allowed to cool. The formation of a
precipitate was observed. Similar procedure was done for the positive control. The results obtained were
compared. For Extons test, 3mL of urine sample and 3mL of Extons reagent were mixed in a test tube.
The solution was warmed until cloudiness appeared. Similar procedure was done for the positive
control. The results obtained were compared. For Smiths test, 5mL urine sample was placed in a test

tube. The test tube was inclined and overlayed with 3mL tincture of alcoholic iodine mixture. Similar
procedure was done for the positive control. The results obtained were compared. For the test for occult
blood, a half spatula guaiac powder was added with 5mL of 95% ethanol in a test tube then mixed. To
this mixture, 5mL hydrogen peroxide was added. Then, 5mL of this solution was added to 3 mL
acidified urine. Similar procedure was done for the positive control. The results obtained were

Results and Discussion

Urea is the major end product of protein nitrogen metabolism in humans. Hepatic enzymes convert
ammonia from amino acids to urea (Harr, 2007). Urea is produced in the liver and excreted through the
kidneys in the urine. The circulating levels of urea depend upon protein intake, protein catabolism and
kidney function. Elevated urea levels can occur with dietary changes, diseases which impair kidney
function, liver diseases, congestive heart failure, diabetes and infections. A positive result shows the
formation of N2 gas or moistening of the sides of the test tubes used. The urea cycle (also known as the
ornithine cycle) is a cycle of biochemical reactions occurring in many animals that produces urea
(NH2)2CO from ammonia (NH3). This cycle was the first metabolic cycle discovered. In mammals, the
urea cycle takes place only in the liver (Wallach, 2007).

Uric acid is an organic compound of carbon, nitrogen, oxygen and hydrogen with the formula C 5H4N4O3.
Xanthine oxidase oxidizes oxypurines such as xanthine and hypoxanthine to uric acid. Uric acid is the
final breakdown product of purine catabolism (Pagana & Pagana, 2002). In most other mammals, the
enzyme uricase further oxidizes uric acid to allantoin. Uric acid is a strong reducing agent and a potent
antioxidant. In humans, about half the oxidant capacity of plasma comes from uric acid. Humans

produce large quantities of uric acid. In human blood, uric acid concentrations between 3.6mg/dL and
8.3mg/dL are considered normal by the American Medical Association, although significantly lower
levels are common in vegetarians due to a decreased intake of purine rich meat (Pagana & Pagana,
2002). Diseases related to elevated uric acid levels are kidney stones, Lesch Nyhan syndrome, CVDs
and diabetes. Uric acid reacts with phosphotungstic acid to produce allantoin and tungsten blue.

H3P W12O40

+ Tungsten Blue

Indican is an indole produced by bacterial action on an amino acid, tryptophan, in the intestine. Most of
the indole is excreted in the feces. The remainder is absorbed, metabolized and excreted as indicant in
the urine. In normal urine, the amount of indican excreted is small. It is increased with high protein diets
or inefficient protein digestion. If not digested properly, or if the wrong types of proteins are ingested,
bowel putrefaction can occur. Problems with protein digestion can be caused by overgrowth of anaerobic
bacteria, intestinal obstruction, stomach cancer, low stomach acid, parasitic infections, malabsorptive
syndromes, fungal infections, lack of digestive enzymes, or liver problems. Following absorption, indole
is converted to 3-hydroxy indole in the liver. Detection of indican in the urine depends upon its
decomposition and subsequent oxidation of indoxyl to indigo blue and its absorption into a chloroform

+ HO

+ Indoxyl Sulfuric Acid K

Creatinine test measures the level of the waste product creatinine in the urine. This test indicates whether
the kidneys are working properly. Creatine is formed when food is changed into energy through a
process called metabolism. Creatine is broken down into another substance called creatinine by the
addition of strong acid or by alkali or by using enzyme, creatine hydroxylase (Guyton & Hall, 2006). If
the kidneys are damaged and cannot work normally, the amount of creatinine in the urine goes down
while its level in the blood goes up. Alkaline picrate solution is composed of saturated picric acid and
10% NaOH. Most methods used for creatinine determination are based upon the Jaffe reaction. The Jaffe
reaction uses saturated picric acid which oxidizes creatinine in alkali forming creatinine picrate, in
which creatinine forms a characteristic orange color when treated with alkaline picrate (Guyton & Hall,

Gunnings test is a test for the presence of ketone bodies in the blood or urine. Ketone bodies are three
water-soluble compounds that are produced as by-products when fatty acids are broken down for energy
in the body. The excess presence of ketones in urine is associated with diabetes or altered carbohydrate
metabolism (Dunning & Fischbach, 2004). Lugols solution consists of 5% iodine and 10% potassium
iodide in 85% distilled water with a total iodine content of 130mg/mL. A positive result shows a
formation of iodoform crystals.

Glucose levels are measured to diagnose diabetes. The normal glucose level in the urine is 180mg/dL.
Urine glucose levels of 300 500mg/dL are common with severe untreated diabetes. Diabetes results

from deficient insulin or decreased sensitivity to insulin. The results of a urine glucose test are abnormal
in cases of renal glycosuria and diabetes mellitus (Pagana & Pagana, 2002).

+ 2Cu


+ 4OH-

Cu O


+ 2H2O

Normally, protein is not found in urine. This is because the kidney is supposed to keep large molecules
in the blood and only filter out smaller impurities. If the kidney is diseased, protein will appear in the
urine. Extons reagent is 5% sulfosalicylic acid in a solution of sodium sulfate. A cloudy solution shows
the presence of albumin.

Hemoglobin breakdown results in bilirubin production. In the liver, bilirubin is conjugated to an acid to
make conjugated bilirubin. Unconjugated bilirubin is water soluble and can be excreted in the urine.
Abnormal bilirubin values may indicate anemia, excessive breakdown of RBC, hepatitis, cirrhosis,
obstruction of biliary duct, toxic liver damage and biliary tree obstruction (Dunning & Fischbach, 2004).
Tincture of alcoholic iodine is usually 10% elemental iodine in ethanol. Addtion od a solution of
alcoholic iodine to urine produces a green color. The green color indicates the presence of bile. The
addition of hydrogen peroxide to 5mL 95% ethanol and guaiac powder oxidizes the guaiac causing a
color change. Heme, a component of hemoglobin found in blood, catalyzes this reaction giving a result
in about two seconds. A blue ring indicates a positive result (Lehmann, 1998).

A. Initial Examination of Urine Sample

Time collected

5:30 am
Dark yellow


B. Qualitative Exam of Normal Organic Constituents


Clear light yellow solution with tiny bubbles on top
Clear light yellow solution
Upper Layer: clear brown solution
Lower Layer: clear light yellow solution
CREATININE Dark orange solution

C. Qualitative Exam for Pathologic Constituents

(ketone bodies)
(bile pigments)

Positive (+) Control

Upper Layer: turbid light yellow
Lower Layer: clear light yellow
Iodoform crystals formed
Milky caramel-colored solution

Urine Sample
Clear yellow solution with
iodoform crystals

Turbid light yellow liquid with

white particles suspended
Light blue green interphase

Clear light yellow solution

Dark blue-green solution

Cloudy blue-green solution

No interphase
Brown solution with light brown

A normal urine specimen should be clear. Cloudy urine may be caused by the appearance of pus, RBCs
or bacterias; however, normal urine also may be cloudy because of ingestion of certain foods.
Abnormally colored urine may also result from a pathologic condition or the ingestion of certain
medicines. Dark yellow urine may indicate the presence of urobilinogen or bilirubin (Pagana & Pagana,

The kidneys assist in acid base balance by reabsorbing sodium and excreting hydrogen. An alkaline
pH is observed in a patient with alkalemia. Bacteria, urinary tract infection, or a diet in citrus fruits or
vegetables may cause increased urine pH. Alkaline urine is associated with calcium carbonate, calcium

phosphate and magnesium phosphate stones. Acidic urine is generally obtained from patients with
academia, which can result from metabolic or respiratory acidosis, starvation, dehydration, or a diet high
in meat products or cranberries. Acidic urine is associated with xanthine, cystine, uric acid and calcium
oxalate stones (Pagana & Pagana, 2002).

Urine pH becomes alkaline on standing, because of the action of urea splitting bacteria, which produce
ammonia. The urine pH of an uncovered specimen will become alkaline because carbon dioxide
vaporizes from the urine. Dietary factors affect urine pH. Ingestion of large quantities of citrus fruits,
dairy products, and vegetables produces alkaline urine, whereas a diet high in meat and certain foods
produces acidic urine (Pagana & Pagana, 2002).

Protein is a sensitive indicator of kidney function. Normally, protein is not present in the urine because
the spaces in the normal glomerular filtrate membrane are too small to allow its passage. If glomerular
membrane is injured, the spaces in the filtrate become larger, and the protein seeps into the filtrate, then
into the urine. If this persists at a significant rate, hypoproteinemia can develop as a result of severe
protein loss through the kidneys. This decreases the normal capillary oncotic pressure that holds fluid
within the vasculature and causes severe interstitial edema (Pagana & Pagana, 2002).

Specific gravity is a measurement of the kidneys ability to concentrate urine (Dunning & Fischbach,
2004). Specific gravity is used to evaluate the concentrating and excretory power of the kidneys. High
specific gravity indicates concentrated urine. Low specific gravity indicates dilute urine. Specific gravity
refers to the weight of the urine compared with that of distilled water. Particles in the urine give it
weight or specific gravity (Tuchman, 2005).

Leukocyte (WBC) esterase is a screening test used to detect leukocytes in the urine. Positive results
indicate urinary tract infection. Leukocyte esterase is nearly 90% accurate in detecting WBC in urine
(Pagana & Pagana, 2002).

Like the leukocyte esterase screen, the nitrite test is a screening test for identification of urinary tract
infection. Nitrite screening enhances the sensitivity of the leukocyte esterase test to detect urinary tract
infection. Nitrite testing is only about 50% accurate in detecting WBCs in the urine (Pagana & Pagana,

Normally, no ketones are present in the urine; however, a patient with poorly controlled diabetes and
hyperglycemia may have a massive fatty acid catabolism. The purpose of this catabolism is to provide
an energy source when glucose cannot be transferred into the cell because of insulin insufficiency.
Ketones are the end products of this fatty acid breakdown (Pagana & Pagana, 2002).

Bilirubin is a major constituent of bile. If bilirubin excretion is inhibited, conjugated hyperbilirubinemia

will result. Bilirubin in urine suggests disease affecting bilirubin metabolism after conjugation or defects
in excretion. For screening, elevated urine bilirubin concentration can indicate previously unsuspected
liver injury due to disease, gallstones or drug toxicity. Bilirubin in the urine will color the urine dark
yellow or orange (Pagana & Pagana, 2002).

It comes to the conclusion that the patient is therefore free form any sickness aside from the fact that the
patient is already close to having diabetes, and should now be watchful of her sugar level intake.

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