D.

KETONE BODIES: LEGALS TEST

OBJECTIVES: TO TEST FOR KETONE BODIES IN THE URINE USING LEGALS
TEST.
PRESENTATION OF RESULTS:
OBSERVATIONS NORMAL URINE
SPECIMEN
ABNORMAL URINE
SPECIMEN
COLOR OF SOLN YELLOW YELLOW
INTERPRETATION
( + / - )
NEGATIVE NEGATIVE

Ketones are substances that are formed when the body breaks down fats and
carbohydrates for energy or food. When there are too many ketones in the body,
they spill over into the urine. Ketones may be found in the urine of people who are
fasting, on starvation diets, or in diabetics who have a very high blood sugar level
because of lack of insulin. Ketones are not present in normal urine.
DISCUSSION OF RESULTS:
A) Why are Ketone bodies not normally detected in urine?
Normally, measurable amounts of ketones do not appear in the urine,
because all metabolize fat is completely broken down into carbon dioxide and
water. However, when the use of available carbohydrates as the major source of
energy becomes compromised, body stores of fat must be metabolized to supply
energy. Ketones are then detected in urine.

B) Name the three ketone bodies appearing in urine and three causes of ketonuria?
Ketone bodies:
a. Acetone
b. Acetoacetic acid
c. Beta-hydroxybutyric acid
Three causes of Ketonuria:
1. Uncontrolled diabetes
2. Glycogen Storage disease
3. Starvation

C) What is the chief source of ketone bodies?
The acetyl-CoA produced by mitochondrial beta-oxidation of fatty acids enters
the Kreb's cycle to produce energy, but that is not the only fate of acetyl-CoA. In
liver mitochondria, some acetyl-CoA is converted to acetoacetate, beta-
hydroxybutyrate, and acetone, collectively called ketone bodies. Ketone bodies
are transported to other tissues such as brain, muscle or heart where they are
converted back to acetyl-CoA to serve as an energy source. What are the
decomposition products of the urine test for ketone bodies.
Acetone may rise spontaneously from acetoacetic acid.

D) What are the decomposition products of the urine test for ketone bodies?
Description Reactant Products
ACETONE will decompose into isobutylene, carbon dioxide and water
Butyric acid will decompose into propylene, carbon monoxide and water

E) Discuss the principle of the sodium nitroprusside reaction, including sensitivity and
possible causes of interference.
Acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to
produce a purple color. The test does not measure beta-hydroxybutyric acid and
is only slightly sensitive to acetone when glycine is also present; however, in as
much as these compounds are derived from acetoacetic acid, their presence can
be assumed and it is not necessary to perform individual tests.



F) If the patient is on low-carbohydrate diet, what advice would you give if a positive
result is given?
Start eating foods with carbohydrates because we need energy in everything that we
do daily.
G) Compare the principle and reaction involved in this test with that of the dipstick
test for ketone bodies.
Ketone tests for urine and blood rely on the Legal reaction, in which
AcetoAcetic acid in a specimen of urine or blood reacts in the presence of alkali
with nitroprusside (nitroferricyanide) to produce a purple-colored complex on a
test strip or a test tablet. The Legal test is semiquantitative; it does not measure
the exact amount of ketones in urine or blood.
Reagent strips currently provide a simple, rapid means for performing
medically significant chemical analysis of urine, including pH, protein, glucose,
ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, and specific gravity. It
consists of chemical-impregnated absorbent pads attached to aplastic strip. A
color-producing chemical reaction takes place when the absorbent pad comes in
contact with urine.
E. UROBILINOGEN AND BILE PIGMENTS: GMELINS TEST
OBJECTIVES: To test for Urobilinogen and bile pigments in the urine sample.
PRESENTATION OF RESULTS:
OBSERVATIONS NORMAL URINE
SPECIMEN
ABNORMAL URINE
SPECIMEN

DISCUSSION OF RESULTS:
1. Outline the steps in the degradation of hemoglobin to bilirubin, urobilinogen and
finally, urobilin.

Bilirubin, a highly pigmented yellow compound, is a degradation product of
hemoglobin. Under normal conditions, the life span of red blood cells is
approximately 120 days, at which time they are destroyed in the spleen and liver
by the phagocytic cells of the reticuloendothelial system. The liberated
hemoglobin is broken down into its component parts: iron, protein, and
COLOR OF RING NO COLORED RING BAND OF COLORED
RING
(RED, GREEN, BLUE,
YELLOW, VIOLET
INTERPRETATION
( + / - )
NEGATIVE POSITIVE
protoporphyrin. The body reuses the iron and protein, and the cells of the
reticuloendothelial system convert the remaining protoporphyrin to bilirubin. The
bilirubin is then released into the circulation, where it binds with albumin and is
transported to the liver. At this point, the kidneys cannot excrete the circulating
bilirubin because not only is it bound to albumin but also it is water insoluble. In
the liver, bilirubin is conjugated with glucuronic acid by the action of glucuronyl
transferase to form water-soluble bilirubin diglucuronide (conjugated bilirubin).
Usually, this conjugated bilirubin will not appear in the urine because it is passed
directly from the liver into the bile duct and on to the intestine. In the intestine,
intestinal bacteria reduce bilirubin to urobilinogen, which is then oxidized and
excreted in the feces in the form of urobilin.

2. State the reasons for increased and decreased urine urobilinogen levels.
Increased Urine Urobilinogen
Increase of production of bilirubin
Excessive destruction of red cells such as in hemolytic anemias, pernicious
anemia, and malaria.
hepatitis, portal cirrhosis, or congestive heart failure
Decreased Urine Urobilinogen
normal amounts of bilirubin is not excreted into the intestinal tract
cholelithiasis, severe inflammatory disease, or neoplastic disease
during antibiotic therapy
normal intestinal flora may prevent conversion of bilirubin to urobilinogen

3. Describe the relationship of urinary bilirubin and urobilinogen to the diagnoses of
the bile duct destruction, liver disease and hemolytic disorders.

4. Compare the principle involved in the procedure performed with the reagent strip
test for urinary bilirubin, including sources of error.
REAGENT STRIP REACTIONS AND INTERFERENCE
Routine testing for urinary bilirubin by reagent strip uses the diazo reaction. Bilirubin
combines with 2,4- dichloroaniline diazonium salt or 2,6-
dichlorobenzenediazonium-tetrafluoroborate in an acid medium to produce an
azodye, with colors ranging from increasing degrees of tan or pink to violet,
respectively. Qualitative results are reported as negative, small, moderate, or
large, or as negative, 1+, 2+, or 3+. Reagent strip color reactions for bilirubin are
more difficult to interpret than other reagent strip
reactions and are easily influenced by other pigments present in the urine. Atypical
color reactions are frequently noted on visual examination and are measured by
automated readers. Further testing should be performed on any questionable
results.
False positive results occurs when:
- Highly pigmented urine are present
- Indican (intestinal disorders)
False Negative results occurs when:
- Specimen is exposed to light
- Ascorbic Acid
- High concentration of nitrites
While in the Gmelin Test, it was used to detect the presence of bile pigments.
In this test, if bilirubin is present, there is formation of various colors at the junction
of the nitric oxide and the sample. The reaction depends on the progressive
oxidation of bilirubin to blue-green biliverdin by the addition of nitric acid, changing
the yellowish brown pigment to blue and purple; then to green color within
seconds (Bruce-Gregorios, 2006). During the stages of oxidation the bilirubin
undergoes a series of changes in color which follow the sequence of the familiar
solar spectrum (Yeo, 2009). Other bile pigments present are also oxidized to
various colored products aside from biliverdin (green) such as: bilicyanin (blue),
bilifuscin (red), and choletelin (yellow) (Rao, 1992).
In this test, positive results may not be seen if the concentration of the bile
pigments is low in urine (Rao, 1992). Bile pigments detected in the urine suggest
jaundice or a liver disease. Gmelin Test is used to determine the presence of
bile pigments in a solution. Positive results would yield the presence of various
colors at the interface of the nitric acid and the sample solution: green, blue, violet,
red, and reddish-yellow.
F. BLOOD: GUIACS TEST
OBJECTIVES: To test for the presence of Blood in the urine sample.
PRESENTATION OF RESULTS:
OBSERVATIONS NORMAL URINE
SPECIMEN
ABNORMAL URINE
SPECIMEN
COLOR OF SOLN YELLOWISH BROWN YELLOWISH BROWN
INTERPRETATION
( + / - )
NEGATIVE NEGATIVE

DISCUSSION OF RESULTS:
1. Differentiate hematuria, hemoglobinuria and myoglobinuria with regard to the
appearance of urine and serum and its clinical significance.
Hematuria - presence of blood in the urine in the form of intact red blood
cell
- Cloudy red urine
- Renal or Genitourinary
Hemoglobinuria- presence of blood in the urine as the product of red blood
cell destruction.
- clear red specimen
- lysis of red blood cells produced in the urinary tract -
Intravascular hemolysis
Myoglobinuria a heme-containing protein found in muscle tissue it results to
muscle destruction(rhabdomyolysis)
- clear red-brown urine
- Cholesterol lowering satin medications.


2. Compare the principle involved in the Guiacs test with the reagent strip method
for blood testing, and list possible causes of interference.

REAGENT STRIP REACTIONS
In the presence of free hemoglobin/myoglobin, uniform color ranging from a
negative yellow through green to a strongly positive green-blue will appear on the
pad. The degree of hematuria can then be estimated by the intensity of the speckled
pattern. REACTION INTERFERENCE
FALSE-POSITIVE
- menstrual contamination
- Strong oxidizing deter- gents are present in the specimen container.
- Vegetable peroxidase and bacterial enzymes, including an Escherichia coli
peroxidase,
- Ascorbic acid
-
While in Guiacs test, is a test for blood in urine or feces using a reagent
containing guaiacum that yields a blue color when blood is present. Some foods
can affect test results. Do not eat the following foods for 3 days before the test:
Red meat
Cantaloupe
Uncooked broccoli
Turnip
Radish
Horseradish
Some medicines may interfere with the test. These include vitamin C, aspirin, and
nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and naproxen.
Ask your doctor or nurse if you need to stop taking these before the test.
Never stop or change your medicine without first talking to your health care
provider.
This test detects blood in the digestive tract. It may be done if:
You are being screened or tested for colon cancer
You have abdominal pain, changes in bowel movements, or weight loss
You have anemia (low blood count)
You say you have blood in the stool or black, tarry stools
A negative test result means that there is no blood in the stool.
Abnormal results may be caused by anything that causes bleeding in the stomach or
intestinal tract, including:
Colon cancer or other gastrointestinal (GI) tumors
Colon polyps
Esophageal varices and portal hypertensive gastropathy
Esophagitis
Gastritis
GI infections
Hemorrhoids
Inflammatory bowel disease
Peptic ulcer
Other causes of positive test may include:
Nosebleed
Coughing up blood and then swallowing it.
3. Discuss methods used to differentiate between hemoglobinuria and
myoglobinuria.
The diagnosis of myoglobinuria usually is based on the patients history and
elevated serum levels of the enzymes creatinine kinase and lactic
dehydrogenase.
The appearance of the patients plasma also can aid in the differentiation. The
kidneys rapidly clear myoglobin from the plasma, leaving normal appearing
plasma, whereas hemoglobin bound to haptoglobin remains in the plasma and
imparts a red color.
The principle of this screening test is based on the fact that the larger
hemoglobin molecules will be precipitated by the ammonium sulfate, and
myoglobin will remain in the supernatant. Therefore, when myoglobin is present,
the supernatant will retain the red color and give a positive reagent strip test for
blood. Conversely, hemoglobin will produce a red precipitate and a supernatant
that tests negative for blood. Myoglobin is not stable in acid urine and, if
denatured, may precipitate with the ammonium sulfate.