Analyzing Urine Metabolism

Metabolism and energy production: analysis of urine
GROUP 2: ALCORDO, AZARCON, BANTILAN, BARTOLO, BERCILLA, DACANAY, SALDANA, RAMAYLA

METABOLISM AND ENERGY PRODUCTION: ANALYSIS OF URINE
• Urine
- liquid byproduct of the body secreted by the kidneys
through a process called urination
• Two unique characteristics of a urine specimen account

for this continued popularity:
1. Urine is a readily available and easily

collected specimen.
2. Urine contains information, which can be
obtained by inexpensive laboratory tests
about many of the body’s major
metabolic functions.
Abnormal Types of Urine
• Proteinuria
• Oliguria
• Polyuria
• Dysuria
• Hematuria
• Glycosuria
A. Color, pH, and Specific Gravity

• Color of urine varies from almost colorless to black.
Major pigments:
• UROCHROME
 is a product of endogenous metabolism
• UROERYTHRIN
 attaches to the urates, producing a pink color to the sediment.
• UROBILIN
 an oxidation product of the normal urinary constituent
urobilinogen imparts an orange-brown color to urine that is not
fresh.

• measurement of how acidic or alkaline a person's urine
is
• the pH of normal random samples can range from 4.5
to 8.0
• Urine under 5.0 is acidic
• Urine higher than 8.0 is alkaline or basic

• density of a solution compared with the density of a similar
volume of distilled water at a similar.
Test for Specific gravity:
• URINOMETRY
 is less accurate than the other methods currently
available and is not recommended by the Clinical and
Laboratory Standards Institute (CLSI) .
• REFRACTOMETRY
 determines the concentration of dissolved particles in a
specimen.
• DIPSTICK
 dipstick test involves holding a piece of specially treated
paper, or litmus paper, in a urine sample.

PRINCIPLE:
• To determine the color, pH and
specific gravity of the urine.
PROCEDURES:
1. Place 5mL of a “Random Urine”, “<
3 y.o Urine”, “Geriatric Urine” and
“Diabetic Urine” samples in separate
test tubes.
2. Describe and record the color of
each specimen as pale, light or dark
yellow, light amber, dark amber,
greenish, brownish, etc.
3. Use pH paper to determine the pH of
the urine samples.
4. Determine the specific gravity of the
urine sample with refractometer.
5. Record all your observations.

Urine Specific
Color pH
Sample Gravity
< 3 years
Dark Yellow Acidic 1.023
old
Geriatric Pale Acidic 1.002
Random Pale Acidic 1.007
Diabetic Dark Yellow Acidic 1.010

B. PROTEIN: BIURET TEST

PRINCIPLE:
• Biuret test is a general test for
compounds having a peptide bond. When
biuret is treated with dilute copper sulfate
in alkaline condition, a purple colored
compound is formed. This is the basis of
biuret test widely used for identification
of proteins and amino acids.
PROCEDURE:
1. Place about 1 mL of each urine sample in
separate test tubes.
2. To each tube, add about 10 drops of 10%
sodium hydroxide solution and mix.
3. Add 5 drops of 10% copper sulfate
solution to each tube, mix and observe
the results.
4. Record your observations.
5. Violet tint= presence of albumin or
related materials in the urine.
B. PROTEIN: BIURET TEST
Urine Sample Result
< 3 years old Negative (-)
Geriatric Negative (-)
Diabetic Negative (-)
Random Negative (-)

Glucose
• Most frequent chemical
analysis performed on urine
• Clinical Significance
• Under normal
circumstances, almost all
the glucose filtered by the
glomerulus is reabsorbed in
the proximal convoluted
tubule
• Renal threshold for glucose
is approximately 160-180
mg/dL
Difference Between Glucose

Oxidase (Reagent Strip)
Reactions and Copper
Reduction Test
Difference Between Glucose Oxidase (Reagent Strip)

Reactions and Copper Reduction Test
• a very specific test for glucose. The testing area of

the strip is a mixture of glucose oxidase, peroxidase,
chromogen and buffer to produce a double sequential
enzyme reaction

• This test relies on the ability of glucose and other
substances to reduce copper sulfate to cuprous oxide
in the presence of akali and heat.

Glucose oxidase reaction
• a very specific test for glucose. The testing area of the strip is a mixture of
glucose oxidase, peroxidase, chromogen and buffer to produce a double
sequential enzyme reaction
Copper Reduction
• This test relies on the ability of glucose and other substances to reduce copper
sulfate to cuprous oxide in the presence of akali and heat.
• Possibleinterference for both these test is

that the urine remained unpreserved at room
temperature for an extended period of time
C. GLUCOSE: BENEDICT’S TEST

PRINCIPLE:
• Benedict’s reagent contains CuSO4.
in the presence of reducing sugars
Cupric Ions are converted to Cuprous
Oxide which is hastened by heating,
to give the color.
PROCEDURES:
1. Place about 1 mL of the “Random
Urine”, “< 3 y.o Urine”, “Geriatric
Urine” and “Diabetic Urine” samples
in separate test tubes.
2. Place 3 mL of Benedict’s solution
in each test tube. Add 5 drops of
urine and mix.
3. Place the test tubes in a boiling
water bath for 2-3 minutes. Let it
stand until cool.
Expected Result Color
(-) Negative No change of color
Green solution with

(+) Positive
yellow precipitate
Yellow solution with
(++)
yellow precipitate
Orange solution with
(+++)
yellow precipitate
Orange to Brick red
(++++)
precipitate
Urine Sample Result
Random Positive (+)

D. KETONE BODIES: LEGAL’S TEST

PRINCIPLE:
• The test for the detection of ketone bodies. The
indication zone contains an alkaline buffer
mixed with sodium nitroprusside, which
gives a purple colored product by a reaction
acetoacetic acid or acetone.
PROCEDURE:
1. Place 1 mL of each sample in separate test
tubes.
2. Add enough NaOH solution to render it slightly
alkaline to litmus paper.
3. Add few drops of Sodium nitroprusside and a
few drops of glacial acetic acid.
4. Observe the resulting change in color of the
solution.
5. Purple or Violet- red color = acetone
6. Red = alcohol or acetic acid and di-acetic
acid
D. KETONE BODIES: LEGAL’S TEST
Urine Sample Result
Random Negative (-)

E. UROBILINOGEN AND BILE PIGMENTS: GMELIN’S TEST

PRINCIPLES:
• Nitric acid oxidizes Bilirubin to
Biliverdin giving different colors
from green to violet.
PROCEDURES:
1. Label 4 tubes “Random Urine”,
“< 3 y.o Urine”, “Geriatric
Urine” and “Diabetic Urine”
samples.
2. Place 3 ml of concentrated
nitric acid in 4 clean and dry
test tubes.

PROCEDURES:
3. Slowly add and let flow 10
drops of urine samples along
the sides of the tube containing
the acid. DO NOT MIX. Note
resulting color formed at the
junction of the two liquids.
4. A band of colored ring at the
point of contact of the two
fluids indicates the presence of
bile pigments. These rings are
GREEN, BLUE, VIOLET, RED,
and YELLOW.
Urine Sample Result
Random Negative (-)

F. BLOOD: GUAIAC TEST (OCCULT BLOOD TEST)

PRINCIPLE:
• based on the oxidation of
phenolic compounds present in
the guaiac to quinones resulting
in production of the blue color.
• Guaiac uses a peroxidase-like
activity found in hemoglobin to
generate an oxidative reaction
with a reagent to produce a blue
color.
PROCEDURES:
1. Label 4 clean and dry test tubes.
2. Mix 1.0 mL of guaiac solution
and 1.0mL of "old oil of
turpentine" together.
PROCEDURES:
3. Layer on this solution about
0.5mL of the urine samples
to be tested. A blue-green ring at
the interface, followed by a light
or dark blue ring will appear at the
point of contact of the two
solutions. If you shake the mixture
well, the entire solution will turn
blue indicating that the test is
positive.
4. Record your results for both a
positive and a negative sample of
urine.
Urine Sample Result
Random Negative (-)

G. ELECTROLYTE: FLAME TEST (SODIUM ION) AND SILVER

NITRATE TEST (CHLORIDE ION)
PROCEDURES:
 FLAME TEST: SODIUM ION
1. Dip cleaned flame test wire (clean in
3M HCl) into each urine specimen.
2. Place the wire loop in a flame
3. Record the color of the flame
 SILVER NITRATE: CHLORIDE ION

1. Place 3mL of each urine specimen in
a separate test tube.
2. Add 5 drops of 3M HNO3 and 5
drops of 0.1M AgNO3. Mix
thoroughly.
3. Note and record the color of the
precipitate formed

Urine Sample Result
< 3 years old Positive (+)
Geriatric Positive (+)
Diabetic Positive (+)
Random Positive (+)


WHAT IS A FLAME TEST? HOW ARE FLAME COLORS PRODUCED?

• The purpose of the flame test is to identify the presence of certain ions in the
sample and in this case, to confirm the presence of sodium ion in the urine.
• In flame test, yellow colouration is produced by the presence of sodium ion in
urine samples. There is no proper standard to specify abnormal or normal
urine sample by measuring the Sodium level in urine.
DISCUSS THE VARIOUS FLAME COLORS OF THE DIFFERENT IONS:

• The colors observed during the flame test result from the excitement of the
electrons caused by the increased temperature.
WHAT IS THE PURPOSE OF DIPPING THE COPPER WIRE INTO THE ACID BEFORE
SUBJECTING IT TO FLAME?
• To make sure that all previous analytes have been removed and that there will
be no interference in the testing of the urine

Urine Sample Result
< 3 years old Positive (+)
Geriatric Positive (+)
Diabetic Positive (+)
Random Positive (+)


WHAT IS THE ROLE OF NITRIC ACID IN THE TEST FOR CHLORIDE?

• It will react with chloride ions to produce a white precipitate.
WHY SHOULD THE SPECIMEN BE ALBUMIN-FREE WHEN PERFORMING THE CHLORIDE

TEST?
• Urine specimen should be free from albumin when performing the chloride
test due to the following reasons:
 Albumin binds with chloride ions
 Albumin has a free thiol group which results in the formation of dimer with
itself and chloride ion
H. DIPSTICK TESTS
PRINCIPLE:
• Reagent strip contains test pads
impregnated with reagent that
specifically react with a test
analyte and register a specific
color change. The change in color
in the pad is then compared to a
comparator chart to determine the
result.
PROCEDURES:
1. Use on samples dipstick test
strip.
2. Dip the strep completely, but
briefly, into the specimen.
3. Compare the reaction colors with
the manufacturer’s chart. Report
your observation.
H. DIPSTICK TESTS
Random Geriatric < 3 y.o Diabetic
Normal (0.1-
Urobilinogen Normal Normal Normal
1)
Glucose Negative Negative Negative 100
Bilirubin + + ++ +
Ketone Negative Negative Negative Negative
Specific
1.025-1.030 1.025-1.030 1.030 1.030
Gravity
Blood Negative Negative Negative Negative
pH 6 6 6 6
Protein Negative Negative Trace Trace
Nitrite Negative Negative Negative Negative
Leukocyte Negative Negative Negative +25
H. DIPSTICK TESTS
REAGENT
SOURCE OF ERROR / INTERFERENCE
STRIP
• Runover from adjacent pads
pH
• Old specimen
False-positive:
• Highly buffered alkaline urine.
• Highly pigmented urine
• Contamination of container with quaternary compound
PROTEIN
• Loss of buffer
• High specific gravity
False-Negative:
• Protein other than albumin
False-positive:
• Contamination by oxidizing agents and detergents
False-negative:
GLUCOSE • High levels of Ascorbic Acid and ketones
• High Specific Gravity
• Low temperature
• Improperly preserved specimen.
H. DIPSTICK TESTS
REAGENT
STRIP
KETONE • Improperly preserved specimen
False-positive:
• Menstrual contamination
• Strong oxidizing agent
BLOOD
• Bacterial peroxidases
False-Negative:
• High specific-gravity/ crenated cells
False-positive:
• Urine pigments
BILIRUBIN • Indican
False-negative:
• Specimen exposure to light
H. DIPSTICK TESTS
REAGENT
STRIP
False-positive:
UROBILINOGEN False-negative:
• Old specimen
• Preservation in formalin
False-positive:
• Improperly preserved specimen
NITRITE False-Negative:
• Insufficient contact time between bacteria and urinary nitrate
• Lack of urinary nitrate
• Large quantities of bacteria converting nitrate to nitrogen
False-positive:
• Strong oxidizing agent
LEUKOCYTE False-negative:
ESTERASE • High concentration of protein, glucose, oxalic acid and ascorbic
acid.
H. DIPSTICK TESTS
Causes of premature deterioration of
reagent strip:
• Moisture
• Volatile Chemical
• Heat
• Light
Deterioration is avoided by:

• Keeping reagent strip in opaque
containers with desiccant
• Removing strips from container just
before testing
• Tightly resealing the bottle
immediately.
• Opening of bottle in volatile fumes
presence is avoided.
• Storing the strips in room
temperature (below 30 degree
Celsiuus)
• Refrigeration is avoided.
CASE ANALYSIS
CASE ANALYSIS
A patient taken to the emergency department after an episode of
syncope has a fasting blood glucose level of 450 mg/dL. Result of
urine analysis are as follow:
COLOR: Yellow
CLARITY: Clear
SP. GRAVITY: 1.015 CREATININE: 200 mg/dL
pH: 5.0 KETONES: 2+
PROTEIN HIGH: 30 mg/dL BLOOD: Negative
GLUCOSE: 250mg/dL BILIRUBIN: Negative
LEUKOCYTES: Negative PROTEIN-LOW: 15mg/dL
NITRITEL: Negative\
a. Explain the correlation between the patient’s blood and urine glucose blood
result.
b. What is the most probable metabolic disorder associated with this patient?
c. Considering the patients condition, what is the significance of the patient’s
protein to creatinine ratio result.
d. If the patient in this study has a normal blood glucose level and normal
protein and creatinine results, to what would be the urinary glucose be
METABOLISM AND ENERGY PRODUCTION: ANALYSIS
OF URINE
GROUP 2: ALCORDO, AZARCON, BANTILAN, BARTOLO, BERCILLA, DACANAY, SALDANA, RAMAYLA
THANK YOU!

Analyzing Urine Metabolism

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Metabolism and energy production: analysis of urine

GROUP 2: ALCORDO, AZARCON, BANTILAN, BARTOLO, BERCILLA, DACANAY, SALDANA, RAMAYLA

• Two unique characteristics of a urine specimen account

1. Urine is a readily available and easily

Abnormal Types of Urine

A. Color, pH, and Specific Gravity

A. Color, pH, and Specific Gravity

A. Color, pH, and Specific Gravity

A. Color, pH, and Specific Gravity

A. Color, pH, and Specific Gravity

Geriatric Pale Acidic 1.002

Random Pale Acidic 1.007

Diabetic Dark Yellow Acidic 1.010

B. PROTEIN: BIURET TEST

B. PROTEIN: BIURET TEST

Urine Sample Result

< 3 years old Negative (-)

Geriatric Negative (-)

Diabetic Negative (-)

Random Negative (-)

Difference Between Glucose

Difference Between Glucose Oxidase (Reagent Strip)

• a very specific test for glucose. The testing area of

Difference Between Glucose Oxidase (Reagent Strip)

Difference Between Glucose Oxidase (Reagent Strip)

• Possibleinterference for both these test is

C. GLUCOSE: BENEDICT’S TEST

C. GLUCOSE: BENEDICT’S TEST

Expected Result Color

(-) Negative No change of color

Green solution with

C. GLUCOSE: BENEDICT’S TEST

Urine Sample Result

< 3 years old Negative (-)

Geriatric Negative (-)

Diabetic Negative (-)

Random Positive (+)

D. KETONE BODIES: LEGAL’S TEST

D. KETONE BODIES: LEGAL’S TEST

Urine Sample Result

< 3 years old Negative (-)

Geriatric Negative (-)

Diabetic Negative (-)

Random Negative (-)

E. UROBILINOGEN AND BILE PIGMENTS: GMELIN’S TEST

E. UROBILINOGEN AND BILE PIGMENTS: GMELIN’S TEST

E. UROBILINOGEN AND BILE PIGMENTS: GMELIN’S TEST

Urine Sample Result

< 3 years old Negative (-)

Geriatric Negative (-)

Diabetic Negative (-)

Random Negative (-)

F. BLOOD: GUAIAC TEST (OCCULT BLOOD TEST)

F. BLOOD: GUAIAC TEST (OCCULT BLOOD TEST)

F. BLOOD: GUAIAC TEST (OCCULT BLOOD TEST)

Urine Sample Result

< 3 years old Negative (-)

Geriatric Negative (-)

Diabetic Negative (-)

Random Negative (-)

G. ELECTROLYTE: FLAME TEST (SODIUM ION) AND SILVER

 SILVER NITRATE: CHLORIDE ION