1) Know the normal constituents of uring (and the processes by which they become part of urine)
2) Identify and understand the significance of abnorma urine components.
3) Define specific gravity and understand the meaning of too high or too low urine specific gravity.
Approximately 150 L of plasma are purified each day by glomerular filtration, tubular secretion, and tubular reabsorption to produce 0.6 to 2.5 L of urine. The amount of urine produced is influenced by environmental temperature, fluid intake, time of day, emotional state, and many other factors.
The composition of urine reveals much about body function. Metabolic waste products such as carbon dioxide, urea, uric acid, creatinine, sodium chloride, and ammonia are normally present and have no particular pathological significance. The presence of albumin (a protein), glucose, ketones, and various other substances, however, may indicate malfunction of the kidneys or some other organ of the body.
In this exercise, you will have an opportunity to do some of the more routine tests that are performed in the analysis of a urine sample. For many of the tests, a Test Strip Method will be employed which utilizes specially prepared reagent test strips. These convenient test strips are designed primarily for patient use and physician office laboratories. The larger clinical laboratories generally use other methods for reasons of economy.
Following a discussion of the normal constituents of urine will be a series of tests to detect the presence of abnormal substances. Students will perform the tests on their own urine sample.
Normal constituents of urine
Normal urine is actually a highly complex aqueous solution of organic and inorganic substances. The majority of the constituents are either waste products of cellular metabolism or products derived directly from certain foods that are eaten. The total amount of solids in a 24-hour urine sample averages around 60 g. Of this total, 35 g are organic and 25 g are inorganic.
The most important organic substances are urea, uric acid and creatinine. Urea is a product formed by the liver from ammonia and carbon dioxide. Ninety-five percent of the nitrogen content of urine is in the form of this substance. Uric acid is an end-product of the oxidation of purines in the body. By weight, there is normally about 60 times as much urea as uric acid in urine. Creatinine is a hydrated form of creatine. There may be twice as much creatinine as uric acid in the urine.
The principle inorganic constituents of urine are chlorides, phosphates, sulfates and ammonia. Sodium chloride is the predominant chloride and makes up about half of the inorganic substances. Since ammonia is toxic to the body and lacking in plasma, there is very little of it normally present in fresh urine. The small amount that is present is probably secreted by nephron tubules. Urine that is allowed to stand at room temperature for 24 hours or longer may give off an odor of ammonia due to the breakdown or urea by bacterial action.
Because of the efficient absorptive properties of renal tubule cells there should be no appreciable amounts of glucose or amino acids in urine. About 0.3 to 1.0 g of glucose in a 24-hour urine sample would be considered normal excretion. Occasionally, higher amounts may occur in individuals during emotional stress.
Abnormal constituents of urine
In determining whether pathological conditions exist through urine analysis, it is necessary to perform both physical and chemical tests. Of the physical tests that are available, only appearance of the urine will be observed. The chemical tests will be for pH, protein, glucose, ketones, hemoglobin. The significance of each abnormality will accompany the specific test.
Materials and Methods
Collection of the Specimen.
The manner of collecting a urine sample is determined by the type of tests to be performed. If a quantitative analysis is to be performed, a 24-hour collection is necessary. When making qualitative tests, random sampling is satisfactory. When looking for pathological substances, it is best to collect urine 3 h after a meal. The first urine collected in the morning is least likely to contain pathological evidence and is usually discarded in 24-hour specimens. Urine should be collected in a clean container and stored in a cool place until tested. In qualitative testing it should be tested 1 to 2 h after voiding. Catheterization is necessary only in bacteriological examinations.
Wear gloves for the collection of urine samples. Handle your own specimen. Empty your bladder into a specimen cup. Record all urine observations and assessments on the Urinalysis Worksheet. Each lab member should test their own sample and record all of their own observations. When you are done with your analysis, flush the urine down the toilet in the rest tooms. Bring the cup back and dispose in the trash box labeled for biological waste.
The first step of any routine urine analysis is the appearance of the urine.Normal urine will vary from light straw to amber in color. The color of normal urine is due to a pigment called urochrome, which is the end-product of hemoglobin breakdown:
Deviations from normal color that have pathological implications are as follows:
1. Milky: pus, bacteria, fat or chyle
2. Reddish amber: urobilinogen or porphyrin. Urobilinogen is produced in the intestine by the action of bacteria on bile pigment. Porphyrin may be evidence of liver cirrhosis, jaundice, Addison's disease and other conditions.
3. Brownish yellow or green: bile pigments. Yellow foam is definite evidence of bile pigments.
4. Red to smoky brown: blood and blood pigments.
Carrots, beets, rhubarb and certain drugs may color the urine, yet have no pathological significance. Carrots may cause increased yellow color due to carotene; beets cause reddening; rhubarb causes urine to become brown.
Evaluate your urine sample according to the above criteria and record your observations on the Urinalysis Worksheet.
B. Transparency (cloudiness or turbidity)
A fresh sample of normal urine should be transparent, but may become cloudy after standing awhile. Cloudy urine may be evidence of phosphates, urates, pus, mucus, bacteria, epithelial cells, fat, and chyle. Phosphates disappear with the addition of dilute acetic acid and urates dissipate with heat. Other causes of turbidity can be analyzed by microscopic examination.
After shaking your sample, determine the degree of cloudiness and record it on the Urinalysis worksheet.
C. Hydrogen Ion Concentration (pH).
Although freshly voided urine is usually acidic (around pH 6), the normal range is between 4.8 and 7.5. The pH will vary with the time of day and diet. Twenty-four-hour specimens are less acidic than fresh specimens and may become alkaline after standing due to bacterial decomposition of urea to ammonia. High acidity is present in acidosis, fevers, and high protein diets. Excess alkalinity may be due to urine retention in the bladder, chronic cystitis, anemia, obstructing gastric ulcers, and alkaline therapy. The simplest way to determine pH is to use pH indicator paper strips.
1. Obtain pH indicator paper strips.
2. Dip the strip into the urine sample and tap the strip on the edge of the urine container to remove excess urine.
3. While the strip is still wet, compare the color with the color chart on the pH strip container to determine the pH.
4. Record your results on the Urinalysis Worksheet.
Although the large size of protein molecules normally prevents their presence in urine, certain conditions can allow them to filter through. Excessive muscular exertion, prolonged cold baths and excessive ingestion of protein may result in physiological albuminuria. Pathologic albuminuria, on the other hand, exists when albumin of the urine is due to kidney congestion, toxemia of pregnancy, febrile disease and anemias.
1. Shake up the sample of urine and dip the test portion of an albustix test strip into the urine. Tap the strip against the edge of the urine container to remove excess urine.
2. Immediately compare the test area with the color chart on the bottle. Note that the color scale runs from yellow (negative) to turquoise (++++).
3. Record your results on the Urinalysis Worksheet.
As stated above, only a small amount of glucose is normally present in urine (0.01 to 0.03 g/100 ml of urine). When urine contains glucose in amounts greater than this, glucosuria exists. This is usually an indication of diabetes mellitus. Lack of insulin production by the pancreas or insensitivity to insulin is the cause of the disease. Insulin is necessary to stimulate the conversion of excess glucose to glycogen in the liver and muscles. It is also essential to stimulate the oxidation of glucose by cells. A deficiency of insulin function, thus, will result in high blood concentrations of glucose. The renal threshold of glucose is around 160 mg/100 ml. Glucosuria indicates that blood concentrations of glucose exceed this amount and the kidneys are unable to accomplish 100% reabsorption of this carbohydrate.
1. Shake up the sample of urine and dip the test portion of a Clinistix test strip into the urine. Remove the strip and tap the strip on the edge of the urine container to remove excess urine.
2. Wait ten seconds and compare the color to the test area with the color chart on the label of the bottle.
3. Note that there are 3 degrees of positivity. The light intensity generally indicates 0.25% or less glucose. The dark intensity indicates 0.5% or more glucose. The medium intensity has no quantitative significance.
4. Record your results on the Urinalysis Worksheet.
Normal catabolism of fats produces carbon dioxide and water as final end products. When there is not an adequate amount of carbohydrate in the diet, or when there is a defect in carbohydrate metabolism, the body begins to utilize an increasing amount of fatty acids. When this increased fat metabolism reaches a certain point, fatty acid utilization becomes incomplete, and intermediary products of fat metabolism occur in the blood and urine. These intermediary substances are the three ketone bodies: acetoacetic acid (diacetic acid), acetone, and beta hydroxybutyric acid. The presence of these substances in urine is called ketonuria.
Diabetes mellitus is the most common disorder in which ketonuria occurs. Progressive diabetic ketosis is the cause of diabetic acidosis, due to the increased concentration of ketoacids which can eventually lead to coma or death. It is for this reason that the detection of ketonuria in diabetics is of great significance.
1. As with the above rapid methods, dip the test portion of a Ketostix test strip into the urine sample, and tap on the edge of the urine container to remove excess urine.
2. Wait fifteen seconds and compare the color of the test strip with the color chart on the bottle label.
3. Record your results on the Urinalysis Worksheet
When red blood cells disintegrate (hemolysis) in the body, hemoglobin is released into the surrounding fluid. If the hemolysis occurs in the blood vessels, the hemoglobin becomes a constituent of plasma. Some of it will be excreted by the kidneys into urine. If the red blood cells enter the urinary tract due to disease or trauma, the cells will hemolyze in the urine. The presence of hemoglobin in urine is called hemoglobinuria.
Hemoglobinuria may be evidence of a variety of pathologies such as hemolytic anemia, transfusion reactions, yellow fever, smallpox, malaria, hepatitis, mushroom poisoning, renal infections, burns, etc.
1. Shake up the sample of urine and dip the test portion of a Hemastix test strip into the urine.
2. Tap the edge of the strip against the edge of the urine container and let dry for 60 seconds.
3. Compare the color of the test strip with the chart on the bottle.
4. Record your results on the Urinalysis Worksheet
H. Specific Gravity.
Urine specific gravity is a measure of urine concentration. It is the weight of a substance, presented as a ratio, compared to an equal volume of water. The specific gravity of a 24-hour specimen of normal urine will be between 1.015 and 1.025. Single urine specimens may range from 1.002 to 1.030. The more solids in solution, the higher will be the specific gravity. The greater the volume of urine in a 24-hour specimen, the lower will be the specific gravity. A low specific gravity will be present in chronic nephritis and diabetes insipidis. A high specific gravity may indicate diabetes mellitus, fever, and acute nephritis.
To measure specific gravity use one of the refractometers. Open the plastic cover on the front of the instrument and place a drop of sample on the glass surface, lower the cover carefully or the sample will splash. While pointing the refractometer toward a light source, look through the eyepiece and adjust the focus ring until the field is clear. You will see a circular field with several numeric scales, bisected by a horizontal line where the upper darker colored portion of the field and the lower lighter colored portion of the field meet. The specific gravity of your sample is the number where this line intersects the UG (urine gravity) scale. Normal urine gravity is between 1.001 and 1.060.
Clean the refractometer by rinsing the sample stage under running tap water and then drying it carefully using lens paper or a tissue.
I. Identification of the unknown
Repeat the above procedures this time using the unknown urine sample provided by your instructor. This sample is premixed by your instructor and is sterile. Record all values on the Urinalysis Worksheet.
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