Urinalysis

Objective:

To explain in a short essay the value of urinalysis information, at the level of 85% proficiency for each student.

In order to achieve this objective, you will need to be able to:

Perform a routine urinalysis on urine.
Measure specific gravity, osmolarity, sodium chloride, protein, glucose, ketones, hemoglobin, bilirubin, and bile salts.

Materials:

Group Supplies:

5 - paper cups for urine collection

1 urinometer

1 eye dropper

1 thermometer

pH paper and dispenser or use the pH meter

1 test tube rack

2 - 4 test tubes

20% potassium chromate solution (in dropper bottle)

2.9% silver nitrate (in dropper bottle)

Labstix Test Strip (leave in stock bottle until you are ready to use it)

Lab Supplies

Vapor Pressure Osmometer

Pipetter with 10 micoliter pipette tips

Concentrated Nitric Acid

powdered sulfur

Lens paper, isopropyl alcohol or Windex

Stock bottle of Lab-stix strips

Safety glasses

100 mL Graduated cylinders

Orange Biological waste disposal bag

Methods:

Using a urine sample, conduct the following tests:

A. Urine Volume: It is necessary to determine the total volume voided. This is conveniently done using a calibrated paper cup to collect the urine. A calibrated cup is one you have prepared ahead of time by adding known volumes of water and marking levels with a grease pencil. Or you may mark the urine level on the cup and determining the volume later using tap water. Please don't use the graduated cylinders for measuring urine directly. You will need about 50 mL of urine for the following tests

Now divide your urine volume by the number of minutes since you last voided. This is your urine formation rate (UFR) in milliliters per minute. Normal urine formation rate is about 1 mL/min.

B1. Specific Gravity: Specific gravity is an expression of the weight of the substance relative to the weight of an equal volume of water. Water, by definition, has a specific gravity of one. The urinometer (hydrometer) includes a float having a scale that indicates directly the specific gravity of the urine. Since the hydrometer is calibrated to read correctly only at 15^oC, it is necessary to correct specific gravity for temperature.

Pour urine into the cylinder of the urinometer until the float is no longer resting on the bottom. Remove any foam with a paper towel.
Making certain that the hydrometer floats freely read the specific gravity to the third decimal place at the meniscus.
Determine the temperature of the urine specimen. Add one unit to the last decimal (thousandth) place of the hydrometer reading for every three degrees above 15^oC. Record your figures on the Data Sheet.
The specific gravity for normal urine is 1.015 - 1.025. The more solids the higher the specific gravity. High values may indicate diabetes mellitus, fever, and acute nephritis or water deprivation. Low values may indicate chronic nephritis, diabetes insipidus or excessive water intake.

B2. Osmolarity: Osmolarity is an expression of the molar concentration of dissolved particles. In human plasma the concentration of dissolved particles is about 290x10^-3 M. In urine the osmolarity can range from about 100 X 10^-3 M to about 1200x10^-3 M. One mole of sodium chloride dissolved in one liter of water will give a solution with a 2 M osmolarity (1 M sodium ions, 1 M chloride ions.)

Osmolarity will be measured with a Vapor-Pressure Osmometer according to instructions given in the lab. Only 10 microliters (µL) of volume is needed for each measurement. Therefore, only a few drops of urine from each sample needs to be saved to measure osmolarity.

C. Appearance: Normal urine is light straw-amber color due to the pigment urochrome, which is the end product of hemoglobin breakdown. If urine is cloudy, a drop of concentrated nitric acid may be added to the urine specimen. This will dissolve the phosphates or calcium and magnesium, which may be the cause of cloudiness, but not pus. Therefore, the use of concentrated nitric acid can be used to distinguish between pyuria (pus in the urine) and a phosphate precipitate.

Observe the following table for color variations of urine and their meaning.

NORMAL AND ABNORMAL VARIATIONS IN URINE COLOR

Color	Cause of Coloration	Pathological Condition
Colorless	Dilution or diminution of pigments	Nervous conditions; Diabetes Insipidus, Hydruria, Granular kidney
Dark yellow to brown red	Increase of normal or pathological pigments	Acute febrile diseases
Milky	Fat globules or pus	Chyluria; Purulent diseases of the urinary tract
Orange to orange red	Excreted drugs	Santonin, Chrysophanic acid, puridium, serenium
Red to reddish	Uroerythrin, uroporphyrin coproporphyrin, myoglobin hemoglobin	Porphyrin, hemorrhages, hemoglobinuria trauma
Brown to brown black	Hematin methemoglobin melanin hydroquinol and catechol	Small hemorrhages methemoglobinuria melanotic sarcoma
Greenish yellow to greenish brown approaching black	Bile pigments	Jaundice
Dirty green or	A dark blue surface scum with a blue deposit, due to an indigo-forming substance	Cholera, typhus; seen especially in urine that has been standing for a few hours

D. Determination of Sodium Chloride Concentration: The normal amount of sodium chloride in urine for a 24-hour period is 15 grams. Using the following procedure, determine the sodium chloride concentration of your specimen:

Measure 10 drops of urine into a test tube using a standard medicine dropper. Add 1 drop of 20% potassium chromate solution to the urine.
Add 2.9% silver Nitrate solution 1 drop at a time using the dropper in the bottle. Vigorously swirl the test tube after each drop of silver nitrate added.
Count the drops of silver nitrate solution required to turn the solution from a bright yellow to an orange brown, color.
Each drop of 2.9% silver nitrate required to produce the color change represents approximately 1 gram/liter of NaCI.

E. Use of Labstix or other Test Strips: Follow the instructions on the bottle containing the Test Strips. Dip the test strip into the urine sample and match the color of the strip with the chart provided on the bottle. Be sure to observe the proper amount of time required for the color to develop. Record data an the sheet provided for each of the following tests:

pH Normal pH ranges from 4.8 to 8.0 but usually urine has a pH value 5.5 - 6.5. High acidity may be due to acidosis, fever, or a high protein diet. Low acidity values may be the result of bladder retention, cystitis, or anemia.
Protein: Normal urine is free of protein although traces of serum albumin and globulin may be present. When proteinuria (protein in the urine) occurs the major type of protein is albumin. The most common cause of proteinuria is renal disease, including toxemia of pregnancy, Albuminuria may also be indicative of ischemic kidneys, congestive heart failure, fever, anemia and liver disease.
Glucose: Glucose is absent in normal urine. The excretion of readily detectable amounts of glucose is known as glucosuria. Glucosuria may be benign or pathological. Renal diabetes is a benign condition where the kidney threshold for glucose is reduced but blood glucose is normal. Diabetes mellitus is a pathological condition where blood glucose shows a marked elevation and urine volume is greatly increased with a glucose content from 3 to 10% or more.
Ketones: Ketones include acetoacetic acid, B-hydroxybutyric acid, and acetone. Such substances are present in urine of individuals who are using body fat as their primary energy source as would occur during starvation, prolonged fasting, or untreated Diabetes Mellitus. Normal urine may contain variable amounts of total ketones (mostly acetone) to the extent of about 20-mg per day. Pathological values for ketones range from 0.02 to 6 grams per day.

Hemoglobin: When blood appears in the urine it may be classified as hematuria or hemoglobinuria. Hematuria consists of red blood cells and pigments in the urine, where hemaglobinuria involves the pigment only present in the urine. Hematuria is caused by lesions in the kidney or urinary tract. Hemoglobinuria results from hemolysis of red blood cells and liberation of hemoglobin. Causes of hemoglobinuria include malaria, typhoid, yellow fever, hepatitis, hemolytic jaundice, transfusion reactions, and bums that cover a considerable amount of the body.
Bilirubin: Normal urine is free of bilirubin. Bilirubin is formed from decomposition of hemoglobin by the reticuloendothelial system and then bound to protein. It is removed from the plasma by the liver and conjugated with glucuronic acid. In this form it is secreted in the bile. Plasma and hence urinary levels of bilirubin increase when there is a biliary obstruction, hepatitis, or liver cirrhosis.
Bile salts: Normally bile salts are not present in the urine, but they may appear due to bilary obstruction which results in a increase of bile acids in circulation. The test used to detect bile salts depends on the ability of bile salts to reduce the surface tension of liquids. The test, called Hay's test, is performed according to the following procedure:

Sprinkle a little finely powdered sulfur upon the surface of cool urine.
If the sulfur sinks at once, bile salts are present in the amount of about 0.01%.
If the sulfur sinks only after gentle agitation, bile salts are present in the amount of 0.0025% or more.
If the sulfur remains floating even after gentle agitation, bile salts are absent.

Results:

DATA SHEET: Urinalysis

Volume Produced	Urine Formation Rate mL/min	Specific Gravity or Osmolarity	Appearance	NaCl conc.

pH	Protein	Glucose	Ketones	Hemoglobin	Bilirubin	Bile salts

Discussion:

What is the value of urinalysis information?
What is glomerular filtration? What is tubular reabsorption? What is tubular secretion? Name 2 substances reabsorbed. Name 2 substances secreted.
Explain the relation between specific gravity and osmolarity.
Explain the relation between osmolarity and sodium chloride concentration.
Name three major waste products from metabolism that are eliminated in the urine via the kidneys.
What is a diuretic? Give and example and explain how it works.