EXPERIMENT 10
Urinalysis
Objectives
1. To perform quick routine analytical tests on urine Samples.
2. To compare results obtained on "Normal" and 'Pathological" urine samples.
Background
The kidney is an important organ that filters materials from the blood that are harmful or in excess or both.
These materials are excreted in the urine. A number of tests are routinely run in clinical laboratories on urine
samples. These involve the measurements of glucose or reducing sugars, ketone bodies, albumin, specific
gravity, and pH.
Normal urine contains little or no glucose, or reducing sugars; the amount varies from 50 to 150 mg/dL.
Higher concentrations may occur if the diet contains a large amount of carbohydrates or strenuous work was
performed shortly before the test. Patients with diabetes or liver damage have chronically elevated glucose
content in the urine. A semiquantitative test of glucose levels can be performed with the aid of test papers
such as Diastix. This is a quick test that uses a paper containing two enzymes: glucose oxidase and
peroxidase. In the presence of glucose the glucose oxidase catalyzes the formation of gluconic acid and
hydrogen peroxide. The hydrogen peroxide is decomposed with the aid of peroxidase and yields atomic
oxygen [O]. The atomic oxygen reacts with an indicator, potassium iodide, producing a red brown color.
The intensity of the color is proportional to the glucose concentration.
!-D-glucose + O 2
H2O 2
peroxidase
glucose
oxidase
gluconic acid + H2O2
H2O + [O]
KI + [O] → red brown oxidation product
Because glucose oxidase exhibits absolute specificity, this test is specific for glucose only. No other reducing
sugar will give positive results.
Normal urine contains no albumin or only a trace amount of it. In case of kidney failure or malfunction the
protein passes through the glomeruli and is not reabsorbed in the tubule. So, albumin and other proteins end
up in the urine. The condition known as proteinuria may be symptomatic of kidney disease. The loss of
albumin and other blood proteins will decrease the osmotic pressure of blood. This allows water to flow from
the blood into the tissues, creating swelling (edema). Renal malfunction is usually accompanied by swelling
of the tissues. The Albustix test is based on the fact that a certain indicator at a certain pH changes its color
in the presence of proteins. Albustix contains the indicator, tetrabromophenol blue, in a citrate buffer at pH
3. At this pH the indicator has a yellow color. In the presence of protein the color changes to green. The
larger the protein concentration, the greener the indicator will be. Therefore, the color produced by the
Albustix can be used to estimate the concentration of protein in urine.
Three substances that are the products of fatty acid catabolism, acetoacetic acid, β-hydroxybutyric acid, and
acetone are commonly called ketone bodies. These are normally present in the blood in small amounts and
can be used as an energy source by the cells. Therefore, no ketone bodies will normally be found in the
urine. However, when fats are the only energy source, excess production of ketone bodies will occur. They
will pass through the filter in the kidney and appear in the urine. Such abnormal conditions of high fat
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catabolism take place during starvation or in diabetes mellitus when glucose, although abundant, cannot pass
through the cell membranes to be utilized inside where it is needed. Acetoacetic acid (CH3COCH2COOH),
and to a lesser extent acetone (CH3COCH3) and β-hydroxybutyric acid (CH3CHOHCH2COOH), react with
sodium nitroprusside (Na2(Fe(CN)3NO))•2H20 to give a maroon-colored complex. In Ketostix the test area
contains sodium nitroprusside and sodium phosphate buffer to provide the proper pH for the reaction. The
addition of lactose to the mixture in the Ketostix enhances the development of the color.
The specific gravity of normal urine may range from 1.008 to 1.030. After excessive fluid intake (a beer
party), the specific gravity may be on the low side; after heavy exercise and perspiration, it may be on the
high side. High specific gravity indicates excessive dissolved solutes in the urine.
The pH of normal urine can vary between 4.7 to 8.0. The usual value is about 6.0. High protein diets and
fever can lower the pH of urine. In severe acidosis the pH maybe as low as 4.0. Vomiting and respiratory or
metabolic alkalosis can raise the pH above 13.0.
Urea in urine may be detected and measured quantitatively by the liberation of ammonia due to the enzymatic
action of urease. The ammonia may be detected by its action on an acid-base indicator like phenol red.
O
H2NCNH 2 + H 2O
urease
2 NH + CO 2
3
Procedure
The stockroom will provide one "normal" and two "pathological" urine samples. While handling body fluids,
such as urine. plastic gloves should be worn. The used body fluid will be collected in a special jar and
disposed collectively. The plastic gloves worn will be collected and autoclaved before disposal. Place 2 mL
of urine sample from each source into three different 10 x 75 mm test tubes. These will be tested with the
different test papers. Record amounts of each substance indicated, not just color observed!
Glucose Test
For the glucose test, take 3 Diastix from the bottle. Replace the cap immediately. Dip the test area of the
Diastix into one of the samples. Tap the edge of the strip against a clean, dry surface to remove the excess
urine. Compare the test area of the strip to the color chart supplied on the bottle exactly 30 sec. after the
wetting. Ignore the color changes that occur after 30 sec. Read the color from blue green (negative) to
colors ranging from green to brown indicating amounts of glucose from 100 up to >2000 mg/dL. Repeat the
test with the other two samples.
.
Protein Test
For the protein test take 3 Albustix strips, one for each of the three urine samples, from the bottle. Replace
the cap immediately. Dip the Albustix strips into the test solutions, making certain that the reagent area of
the strip is completely immersed. Tap the edge of the strip against a clean, dry surface to remove the excess
urine. Compare the color of the Albustix test area with the color chart supplied on the bottle. The time of the
comparison is not critical, you can do it immediately or any time within a minute after the wetting. Read
the color from yellow (negative) to different shades of green, indicating trace amounts of albumin up to
>2000 mg/dL.
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Ketone Bodies
To measure the ketone bodies' concentration in the urine, take 3 Ketostix strips from the bottle. Replace the
cap immediately. Dip a Ketostix, one in each of the urine samples, and remove the strips at once. Tap them
against a clean, dry surface to remove the excess urine. Compare the color of the test area of the strips to the
color chart supplied on the bottle. Read the colors exactly 15 sec. after the wetting. A buff pink color
indicates the absence of ketone bodies. A progression to a maroon color indicates increasing concentration of
ketone bodies from 5 to 160 mg/dL of urine.
pH in Urine
To measure the pH of each urine sample, use pH indicator paper, such as pHydrion test paper within the pH
range of 4.0 to 10.0. Dip stick into the sample and compare to color chart while still moist.
Specific Gravity of Urine
The specific gravity of a urine sample will be measured with the aid of a urinometer (see Fig. 9.1). Place the
bulb in a cylinder. Add sufficient urine to the cylinder to make the bulb float. Read the specific gravity of
the sample from the stem of the urinometer where the meniscus of the urine intersects the calibration lines.
Be sure the urinometer is freely floating and does not touch the walls of the cylinder. Your reading should
be accurate to three decimal places.
Urea in Urine (Do this test first and let it react while completing the rest of the analyses. )
To 5 mL of normal urine only, add 3 drops of phenol red solution. If the solution is pink, add one or two
drops of dilute acetic acid until it turns yellow. Then add 2 mL of a fresh 1% urease solution and mix well.
Look for a color change.
Figure 9.1 A urinometer
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Chemicals and Equipment
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Diastix (36 /lab)
Albustix (36 /lab)
Ketostix (36 /lab)
pH paper in the 4.0 to 10.0 range (36/lab)
urinometer (3)
10 x 75 mm test tubes
1% urease (25 ml/lab)
phenol red
urine samples: normal A (100 ml/lab) , pathological B (30 ml/lab), and pathological C (30 ml/lab)
dilute acetic acid
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NAME__________________________
SECTION__________
PARTNER_______________________
DATE_____________
Experiment 9: Urinalysis
DATA SHEET
Urine Samples
Test
Normal A
Pathological B
Pathological C_____
Glucose _________________________________________________________________________
Ketone
Bodies
_________________________________________________________________________
Protein
(albumin)
_________________________________________________________________________
pH
_________________________________________________________________________
Specific
gravity
_________________________________________________________________________
Urea
_________________________________________________________________________
Make a diagnosis of pathological urine sample B and explain your reasoning:
Make a diagnosis of pathological urine sample C and explain your reasoning:
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POST-LAB QUESTIONS
1. In determining glucose concentration in urine you use a Diastix. Yet the color of a positive test is the
result of the interaction of a potassium iodide indicator with atomic oxygen. How is atomic oxygen
related to glucose?
2. Urine samples came from three different persons. Each had a different diet for a week. One lived on a
high protein diet; the second, on a high carbohydrate diet; and the third lived on a high fat (no
carbohydrate) diet. How would their urine analyses differ from each other? (Compare pH, specific
gravity, presence of glucose and/or ketone bodies, and quantity of urea.)
3. A patient's urine was tested with Diastix and the color was read 60 seconds after wetting the strip. It
showed 1000 mg/dL glucose in the urine. Is the patient diabetic? Explain.
4. Under what conditions might glucose be excreted in the urine?
5. If a urine sample from a patient gave a strongly positive test for both glucose and ketone bodies, what
diagnosis might be made? Explain.
6. Why is albumin not normally present in the urine?
7. What pathological conditions result in the excretion of albumin in the urine?
8. How could a person alter the quantity of urea excreted in 24 hours?
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