28TH SEPTEMBER 2022.

URINALYSIS: THE PHYSICAL AND CHEMICAL

EXAMINATION OF URINE.

NAME OF GROUP REGISTRATION NUMBER

MEMBER
AYEBAZIBWE CHRISTIANA 21/U/05703/PS
BAGONZA MARK JUNIOR 21/U/09937/PS
LUBEGA FRED 21/U/1444
ABSTRACT:
Urinalysis is a test used to determine the physical and chemical characteristics of a particular urine
sample in order to diagnose disease and to monitor progression of treatment in patients. In this
experiment, urinalysis was carried out on three different urine samples labelled N, T and C. Sample N
was obtained from a normal student while samples T and C were obtained from patients. It was
found that sample N was a normal sample, sample T was from a person with ______ and sample C
was from a person with______ (diabetes-related ketoacidosis).

INTRODUCTION:
Urine is very important in being able to determine the functioning of the body of a human being and
therefore urinalysis is a very important diagnostic tool in hospitals. Urinalysis is a test, requiring
about 10 to 15 milliliters of urine, that is used to determine the physical and chemical characteristics
of a urine sample. The physical characteristics that are tested for are color, foam, smell,
transparency and specific gravity. The chemical characteristics that are tested for are pH and
presence of substances like glucose, ketone bodies, protein, bilirubin and urobilinogen. A deviation
in the norm of any of these parameters may indicate that there is an abnormality in the body and
further evaluation needs to be done because urinalysis alone cannot be used to establish an
abnormality in the body. Urinalysis can only be used to give a general overview of the person’s
health. Results from urinalysis are used together with the symptoms presented and the laboratory
tests in order to pin down the abnormality in the body. It should also be noted that a normal urine
sample does not necessarily indicate that the person is healthy as some people will not release
elevated amounts of substance early in a disease process, some people will release substances in the
urine during the day which might be missed by a single urine sample an in dilute urine, small
quantities of chemicals might be undetected. This is why it is essential to carry out other diagnostic
tests as urinalysis gives a general overview.

Aim of the experiment:

To determine the physical and chemical properties of each of the urine samples that are provided.

To establish the relationship between the physical properties and the chemical properties of each
urine sample.

Significance:

To be able to use information got from urinalysis to diagnose different body abnormalities and
monitor progress in patients on treatment.

BACKGROUND:
A urinalysis is a common test that can assess many different aspects of your health with a urine of
energy sample. Urine is formed by three main processes; glomerular filtration, tubular reabsorption
and tubular secretion. These processes occur in the nephrons. The urine formed provides the health
workers with an idea of the body’s metabolism; whether it is going on well or not. Certain
substances are not supposed to be found in urine and if they are found in urine, that indicates that
there is an issue with the metabolism depending on the substance that is found in the urine.
Glucose, for example, is not supposed to be found in urine as all the glucose is supposed to be
reabsorbed in the body since the body uses glucose as a first priority metabolite for energy
production. If glucose is found in the urine, it is likely that the person has diabetes. Therefore, using
the physical and chemical properties of urine, we can be able to obtain an idea of the body
metabolism of the person whose urine sample was provided.

The physical properties of urine are the first to be observed. The physical properties provide us with
preliminary information which is then confirmed with the chemical tests. For example, if a sample of
urine has a greenish color, that gives us an idea that bile pigments are present in the urine. However,
a chemical test also has to be done in order to confirm the presence of the bile pigments. The
physical properties include color, transparency, smell, foam and specific gravity.

Color:

The color of urine is determined by the concentration of these pigments; urochrome and to a lesser
extent, urobilin and uroerythrin. The color of normal urine may vary from pale yellow to dark amber.
Very pale or colorless urine is very dilute and it can indicate recent fluid consumption, presence of
diuretics and illnesses such as polyuria, diabetes mellitus and diabetes insipidus. An abnormal urine
color can also be caused by presence of blood in the urine causing the urine to have a red color but
the urine color can also vary from pink to black. The final color is usually determined by the amount
of red blood cells present in the urine, the length of contact between the urine and the pigment and
the pH of the urine. For example, acidic urine containing hemoglobin will turn brown after sometime
due to oxidation of hemoglobin to methemoglobin. Presence of proteins like hemoglobin and
myoglobin also cause the urine to turn red indicating hemoglobinuria and myoglobinuria
respectively. Brown or black urine on standing may also indicate presence of malignant melanoma or
alkaptonuria. It should be noted that there are non-pathogenic causes of red urine such as ingestion
of pigmented foods like beetroot, menstrual contamination and some medications. Blue/green urine
color can be caused by bacterial infections.

Transparency:

Normal urine is usually clear but it might become cloudy due to precipitation of amorphous
phosphates in alkaline urine or amorphous urates in acidic urine. Particularly from specimens from
women, urine can be hazy due to presence of mucus and squamous epithelial cells but the urine is
still normal. A cloudy urine sample is abnormal and it is commonly an indicator of presence of
leukocytes, erythrocytes and bacteria.

Smell:

Normal urine has an aromatic smell when it has just been ejected from the body. As it continues to
stand, the urea in the urine is broken down to form ammonia causing an odor of ammonia which
becomes prominent with time.
Below is a table that shows the common causes of different urine odors.

Odor Cause
Aromatic Normal

Foul, ammonia-like Bacterial decomposition, urinary tract

infection
Fruity, sweet Ketones (diabetes mellitus, starvation,
vomiting)
Maple syrup Maple syrup urine disease

Mousy Phenylketonuria

Rancid Tyrosinema

Sweaty feet Isovaleric acidemia

Cabbage Methionine malabsorption

Bleach Contamination

(Adapted from Urinalysis and Body Fluids (5th ed., p. 49) by Susan King Strasinger and Marjorie
Schaub Di Lorenzo, 2008 by F.A. Davis Company.)

Foam:

Normal urine forms a small amount of foam after agitation. For urine that has moderate or large
amount of protein, a white stable foam is formed upon agitation. If there is sufficient amount of
bilirubin in the urine sample, the foam that is formed may appear yellow to yellow-green.

Specific gravity:

Specific gravity is the ratio of the mass of volume of urine to the mass of an equal volume of distilled
water. The specific gravity of urine can be used to measure the concentrating and diluting ability of
the kidney. It can be determined using tools like a urinometer and refractometer, specific gravity
reagent strips and harmonic oscillation densitometry. For a urine sample to be qualified as normal,
the specific gravity should be in the range of 1.015 to 1.045. A low specific gravity in accordance with
that range is caused by dehydration, lipid nephrosis, proteinuria, eclampsia, renal stenosis, to
mention but a few. A high specific gravity in accordance with the range is caused by diabetes
insipidus, collage disease, protein malnutrition, pyelonephritis and other disorders.

The chemical properties are then observed through different reactions particular to different
substances. The chemical analysis is necessary in order to confirm what we observed in the physical
tests. Chemical tests include tests for pH, glucose, ketone bodies, protein, bilirubin and urobilinogen.
pH:

pH is the measure of hydrogen ion concentration in a solution. It is the negative logarithm of the
hydrogen ion concentration. pH is a very important test because it enables one to have an idea on
the working of the kidneys in terms of maintaining an acid-base balance in the body which is a very
important role played by the kidney in conjunction with the lungs. This is done by secretion of
protons and reabsorption of bicarbonate ions. pH does not have a normal range as it keeps varying
from time to time and therefore it is important to consider the urine information with other patient
information like the patient’s renal function, acid-base content of the blood, presence of a urinary
tract infection and other causes. pH can be measured using test strips.

Protein:

During glomerular filtration, most of the protein in blood does not cross the filtration barrier and the
protein which manages to pass through the filtration barrier is reabsorbed in the tubules. Therefore,
in normal urine, a very small amount of protein is present. Proteins that can be found in a normal
urine sample include Tamm-Horsfall proteins which are secreted by the renal tubules, albumin in
very small amounts, serum and tubular microglobulins and proteins from prostatic, seminal and
vaginal secretions. Proteinuria, an excess of protein in urine, can be grouped into three categories
depending on the source of the protein. The three categories are prerenal proteinuria, renal
proteinuria and postrenal proteinuria. Proteinuria can be an indicator of renal disease but it is not a
confirmation of renal disease. Presence of protein in the urine requires additional testing to
determine whether the presence of the protein represents abnormal or pathological condition.
Prerenal proteinuria can be caused by hemoglobinuria, myoglobinuria and multiple myeloma which
causes elevated levels of Bence Jones proteins. Renal proteinuria can be caused by nephritis,
nephrotic syndrome, Fanconi syndrome and any condition that leads to glomerular or tubular
damage. Renal proteinuria can also occur due to non-pathological causes like strenuous exercise,
high fever, exposure to cold and dehydration. Postrenal proteinuria can be caused by bacterial
infections, fungal infections, inflammation, blood due to injury or menstrual contamination, prostatic
fluid and large amounts of spermatozoa.

Protein in urine can be detected using the sulphosalicylic acid turbidity test. In this test, 6%
sulphosalicylic acid is used. The principle behind this experiment is that the protein will be
precipitated by the acid. In presence of protein, a turbid solution will form and the more the protein
concentration, the more the flocculation. It is important to note that there are some precautions
that should be taken before the test is carried out in order to provide accurate results. First of all, in
case the urine is highly alkaline, a more concentrated solution of sulphosalicylic acid can be used or
the pH of the urine sample can be reduced to around pH of 6 by use of acetic acid. Secondly, the
urine sample should undergo centrifugation to remove extraneous contamination. If the urine is
cloudy, it should be filtered first. This test can be used for all forms of protein.

Reagent strips can also be used to determine presence of protein in urine. This colorimetric method
used in dipsticks is based on the concept known as the “protein error of indicators,” a phenomenon
which means that the point of colour change of some pH indicators is different in the presence of
protein from that observed in the absence of protein, because proteins act as hydrogen ion
acceptors at a constant pH. Usually, the indicator changes from yellow to blue (or green) between
pH 3 and pH 4, but in the presence of protein, this colour change will occur between pH 2 and pH 3.
Therefore, in the presence of protein an “error” occurs in the behaviour of the indicator. It is
important to note that reagent strips are mainly sensitive to albumin and less sensitive to globulins
and other proteins and so a negative dipstick test does not necessarily rule out presence of proteins
in the urine. Depending on the manufacturer, the protein area of the strip contains either
tetrabromphenol blue or 3′, 3′′, 5′, 5′′-tetrachlorophenol-3, 4, 5, 6-tetrabromosulfonphthalein and an
acid buffer to maintain the pH at a constant level. At a pH level of 3, both indicators appear yellow in
the absence of protein; however, as the protein concentration increases, the colour progresses
through various shades of green and finally to blue. Readings are reported in terms of negative,
trace, 1+, 2+, 3+, and 4+; or the semiquantitative values of 30, 100, 300, or 2000 mg/dL
corresponding to each colour change. Trace values are considered to be less than 30 mg/dL.
Interpretation of trace readings can be difficult. Reporting of trace values may be a laboratory
option. The specific gravity of the specimen should be considered because a trace protein in a dilute
specimen is more significant than in a concentrated specimen.

Glucose:

Under normal circumstances, all the glucose that is part of the glomerular filtrate is actively
reabsorbed to maintain glucose concentration in the body. When there are elevated levels of
glucose in blood, tubular reabsorption of glucose ceases because the renal threshold for glucose,
which is 160 to 180 mg/dL, has been reached and now glucose will appear in the urine and this can
be termed as glycosuria. A normal person can also have glycosuria after eating a meal very rich in
carbohydrate. Hence, the best time to do an analysis for glucose in urine is after a patient has fasted
for about two hours prior to sample collection. Glycosuria can be an indication of diabetes,
pancreatitis, pancreatic cancer, acromegaly, Cushing’s syndrome, hyperthyroidism, end-stage renal
disease, cystinosis and Fanconi syndrome.

Urinary glucose can be measured by the reagent strip reactions which utilize glucose oxidase enzyme
and hence this method is particular to glucose. This method works on the principle that when
glucose reacts with oxygen in presence of glucose oxidase, hydrogen peroxide and gluconic acid will
be formed. The hydrogen peroxide formed will then react with the chromogen on the strip and the
chromogen will be oxidized causing a color change and the color change will be compared with
colors on a glucose chart provided by the manufacturer to obtain the results. The results should be
obtained at 30 or 60 seconds in order to obtain accurate results. Any results obtained beyond the
stipulated time are irrelevant.

It can also be measured by Benedict’s test which is based on the principle that glucose or other
reducing substances will reduce copper sulphate to cuprous oxide in presence of alkali and heat. A
color change progressing from blue through green, yellow, and orange/red occurs when the reaction
takes place. The progressive color changes indicate how much reducing sugars are present that is
the more the color change, the more the reducing sugars present. The Clinitest tablet which employs
Benedict’s principle can also be used. The tablet contains copper sulphate, sodium carbonate,
sodium citrate, and sodium hydroxide. Upon addition of the tablet to water and urine, heat is
produced by the hydrolysis of sodium hydroxide and its reaction with sodium citrate, and carbon
dioxide is released from the sodium carbonate to prevent room air from interfering with the
reduction reaction. Much as this test is not specific to glucose, it is an important test to carry out
especially in infants as it enables early detection of metabolic disorders that are characterized by
excretion of reducing sugars for example in galactosemia where the urine has galactose. This test
cannot work as a confirmatory test for glucose in urine since it is not specific to glucose.

Ketone bodies:

Ketones or ketone bodies are formed from the liver from acetyl coenzyme A and they can be used to
provide energy. The source of acetyl coenzyme A is catabolism of excess fatty acids. There are three
examples of ketones; acetone, acetoacetate and β-hydroxybutyrate. Acetone, however, is not used
for energy as it cannot be metabolized and it is readily exhaled. During conditions like diabetes
mellitus or starvation, ketones are produced from excess fatty acids in very high amounts that
cannot be utilized by extra hepatic tissues and so, they accumulate in the blood and are later
excreted in the urine, a condition termed as ketonuria. Increased fat metabolism which in turn leads
to ketonuria is caused by inability to metabolize carbohydrates like in diabetes mellitus, increased
loss of carbohydrate from vomiting and inadequate uptake of carbohydrate which can be either due
to starvation or malabsorption. Testing for ketone bodies is very important in management and
monitoring of patients with insulin-dependent diabetes mellitus. In an instance that such a patient
has ketonuria, it indicates that the dosage has to be regulated since there is an insulin deficiency.

Ketones can be detected in urine by carrying out Rothera’s test. The principle of this test is that
acetoacetate and acetone will react with an alkaline solution of sodium nitroprusside (a constituent
of Rothera’s powder) to form a purple colored complex. Β-hydroxybutyrate does not react in this
test.

Bilirubin:

Bilirubin is a highly pigmented yellow compound that is formed from breakdown of hemoglobin in
the reticuloendothelial system. Bilirubin enters the circulation and binds to albumin and is
transported to the liver where it is conjugated with glucuronic acid to form bilirubin diglucuronide
which is also known as conjugated bilirubin. Conjugated bilirubin moves from the liver through the
bile duct into the intestine. Therefore, conjugated bilirubin does not enter the circulation in normal
circumstances. In cases of liver damage or obstruction of the bile duct, conjugated bilirubin will
appear in the blood and it will be a part of the glomerular filtrate since it is not attached to proteins
and eventually, it will be part of the urine. Analysis of bilirubin in urine can also help to establish the
type of jaundice that a patient has. There are three types of jaundice; pre-hepatic jaundice, hepatic
jaundice and post-hepatic jaundice. In hepatic jaundice and post-hepatic jaundice, there are
significant levels of conjugated bilirubin in the serum which can then be detected in the urine while
in pre-hepatic jaundice, there are elevated levels of unconjugated bilirubin in the serum.
Unconjugated bilirubin cannot be detected in urine because it is bound to albumin in the blood
circulation and it is also insoluble in water and so the kidneys cannot excrete it.

Bilirubin can be tested for by use of Fouchet’s test. Fouchet’s reagent is used in this test and it is
composed of trichloroacetic acid, distilled water and 10% ferric chloride. The principle behind this
test is that bilirubin will react with ferric chloride in acid solution to give a green color which
confirms the presence of the bilirubin pigment. In a normal urine sample, there is no bilirubin that is
present.

Urobilinogen:

When conjugated bilirubin is excreted through the bile duct into the intestine, the intestinal bacteria
convert the bilirubin to a combination of urobilinogen and stercobilinogen. Some of the urobilinogen
is reabsorbed from the intestine into the blood, recirculates to the liver, and is excreted back into
the intestine through the bile duct. The stercobilinogen cannot be reabsorbed and remains in the
intestine where it is oxidized to urobilin and excreted in the faeces. Urobilin is the pigment
responsible for the characteristic brown colour of faeces. Urobilinogen appears in the urine because,
as it circulates in the blood en route to the liver, it passes through the kidney and is filtered by the
glomerulus. Therefore, a small amount of urobilinogen— less than 1 mg/dL or Ehrlich unit—is
normally found in the urine. Increased urine urobilinogen (greater than 1 mg/dL) is seen in liver
disease and haemolytic disorders. Impairment of liver function, for example in hepatitis and
cirrhosis, decreases the ability of the liver to process the urobilinogen recirculated from the
intestine. The excess urobilinogen remaining in the blood is filtered by the kidneys and appears in
the urine. The clinical jaundice associated with haemolytic disorders results from the increased
amount of circulating unconjugated bilirubin. This unconjugated bilirubin is presented to the liver for
conjugation, resulting in a markedly increased amount of conjugated bilirubin entering the
intestines. As a result, increased urobilinogen is produced, and increased amounts of urobilinogen
are reabsorbed into the blood and circulated through the kidneys where filtration takes place. In
addition, the overworked liver does not process the reabsorbed urobilinogen as efficiently, and
additional urobilinogen is presented for urinary excretion. The absence of urobilinogen in the urine
and faeces is also diagnostically significant and represents an obstruction of the bile duct that
prevents the normal passage of bilirubin into the intestine. In diarrhea, absorption of urobilinogen
from the gut is reduced. In polyuria, concentration of urobilinogen will be low.

Urobilinogen can be tested for in urine by Ehrlich’s test. In this test, Ehrlich’s reagent, which contains
p-dimethylaminobenzaldehyde, is used. The principle of the reaction is that urobilinogen reacts with
benzaldehyde to give a red color. For normal urine, the color given is pink to light red indicating a
small amount of urobilinogen and for abnormal urine, it either has no pink color indicating no
urobilinogen or it has a deep red color indicating increased urobilinogen.