Urine Analysis Lab Con

D. KETONE BODIES: LEGAL’S TEST OBJECTIVES: TO TEST FOR KETONE BODIES IN THE URINE USING LEGAL’S TEST. PRESENTATION OF RESULTS: OBSERVATIONS

NORMAL

URINE ABNORMAL

SPECIMEN

URINE

SPECIMEN

COLOR OF SOL’N

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, betahydroxybutyrate, 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 OBSERVATIONS

NORMAL SPECIMEN

URINE ABNORMAL SPECIMEN

URINE

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: GMELIN’S TEST OBJECTIVES: To test for Urobilinogen and bile pigments in the urine sample. PRESENTATION OF RESULTS:

COLOR OF RING

NO COLORED RING

BAND OF RING

COLORED

(RED, GREEN, BLUE, YELLOW, VIOLET

INTERPRETATION

NEGATIVE

POSITIVE

(+/-)

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

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: GUIAC’S TEST OBJECTIVES: To test for the presence of Blood in the urine sample. PRESENTATION OF RESULTS: OBSERVATIONS

NORMAL

URINE ABNORMAL

SPECIMEN COLOR OF SOL’N

YELLOWISH BROWN

URINE

SPECIMEN 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 - clear red-brown urine - Cholesterol lowering satin medications.

destruction(rhabdomyolysis)

2. Compare the principle involved in the Guiac’s 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 Guiac’s 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 patient’s history and elevated

serum

levels

of

the

enzymes

creatinine

kinase

and

lactic

dehydrogenase. The appearance of the patient’s 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.