HSBTE DMLT Urine Analysis: Normal composition of urine, Qualitative analysis of proteins, sugar, ketone bodies, bilirubin, and blood. Detailed discussion on glycosuria and albuminuria. Clinical significance of urine analysis.

Normal composition of urine

The normal composition of urine reflects the body's waste elimination process and provides information about the functioning of the kidneys. The typical components of normal urine include

• 1. Water: Water constitutes about 95% of the total volume of urine. Adequate hydration levels influence the color and concentration of urine.

• 2. Urea: Urea is a byproduct of protein metabolism. Urea is the main nitrogenous waste in the urine. It contributes to the characteristic odor of urine.

• 3. Uric Acid: This compound is a byproduct of the breakdown of purines. These are substances found in certain foods. Elevated levels may indicate conditions like gout.

• 4. Ammonia: Ammonia is a product of protein metabolism and is usually present in small amounts in urine.

• 5. Creatinine: Creatinine is a waste product generated from the breakdown of muscle tissue. Its levels in urine can provide information about kidney function.

• 6. Creatine: A breakdown product of creatine phosphate, which is involved in energy metabolism in muscles.

• 7. Electrolytes:

  • · Sodium (Na): Essential for maintaining fluid balance.

  • · Potassium: Important for nerve and muscle function.

  • · Chloride: Plays a role in maintaining electrolyte balance.

• 8. Bicarbonate (HCO3): Maintain the pH balance of urine.

9. Enzymes: Only trace amounts of enzymes are found in urine. Elevated levels indicate damage to the urinary tract or other organs.

• 10. Hormones: Small amounts of hormones may be excreted in urine. It reflects the endocrine system's activity.

• The specific composition of urine depends on factors such as diet, hydration status, and individual health. Abnormal levels of certain substances in urine may indicate underlying health conditions. Interpretation of urine analysis results requires consideration of the overall clinical context.

Qualitative analysis of proteins, sugar, ketone bodies, bilirubin, and blood.

Qualitative analysis of proteins, sugar, ketone bodies, bilirubin, and blood in urine assesses the presence or absence of these substances. It does not measure their exact concentrations. These tests provide valuable information about various health conditions and are typically part of a urinalysis.

Qualitative analysis of proteins in urine.

Urine contains only a trace amount of protein in normal conditions. An elevated level of protein in urine is called proteinurea. Proteinuria indicates kidney dysfunction or kidney damage. Conditions such as glomerulonephritis, nephrotic syndrome, or kidney infection can lead to increased protein excretion.

The following methods are used for qualitative analysis of proteins in urine.

• Urine Dipstick test

  • 1. Collection of urine: A midstream urine sample is collected from the patient. (midstream: allowing the initial portion of urine to be rejected before collecting the sample). In some cases, a 24-hour urine collection may be required for specific tests.

  • 2. Dipstick Test: Primarily detects albumin. It is the most common method for qualitative analysis of protein in urine. A urine dipstick is a thin, plastic strip with pads or reagent areas containing specific chemicals. The dipstick is dipped into the urine sample. The color changes on the pads indicate the presence or absence of protein in urine. The color changes on the protein pad are compared to a color chart provided by the manufacturer. The chart indicates the degree of protein concentration in the urine. The color intensity may be categorized as negative, trace, 1+, 2+, 3+, or 4+. The higher numbers indicate an increase in levels of protein in urine.

• Sulfosalicylic acid (SSA) test:

  • The urine sample is centrifuged to get a clear supernatant. Take 2 mL of clear urine in a test tube. Add an equal volume of 3% sulfosalicylic acid solution. Mix and allow to stand for 10 minutes. This is a semi-quantitative test. Cloudiness in the sample is compared with a standard test tube containing a known concentration of protein. Trichloroacetic acid may be used in place of sulfosalicylic acid.

• Principle: Urine contains soluble protein. Acid, such as sulfosalicylic acid or Trichloroacetic acid denaturate soluble proteins. Protein denaturation causes precipitation of protein. Protein precipitate develops cloudiness in the urine.

• Heat and Acetic Acid test:

  • Make the urine sample acidic by adding a few drops of 3% acetic acid. Centrifuge the acidic urine and collect the supernatant clear portion of urine. Transfer urine into a clean and clear test tube. Heat the upper portion of the test tube. A white cloud appears in the hot portion of the test tube. The white cloud is due to protein or phosphate in the urine. Add 2 or 3% glacial acetic acid. If the cloud disappears, it is due to phosphate. If cloud is present it is due to albumin.

• Clinical Significance:

  • 1. Kidney Disease: Such as glomerulonephritis, nephrotic syndrome, or diabetic nephropathy.

  • 2. Urinary Tract Infection: Infections may cause temporary increases in protein levels

  • 3. Hypertension (High Blood Pressure): Can contribute to kidney damage and proteinuria.

  • 4. Other Systemic Diseases: Conditions like systemic lupus erythematosus (SLE) or multiple myeloma.

Qualitative analysis of sugar

Qualitative analysis of sugar in urine is a diagnostic test used to detect the presence of glucose (sugar) in urine. This test is often performed as a part of routine urinalysis or to screen for conditions such as diabetes mellitus.

• 1. Sample Collection: collect a clean-catch or midstream sample to avoid contamination.

• 2. Test Strips: Test strips containing specific reagents are used for the qualitative analysis of sugar in urine. These strips usually contain a combination of enzymes, such as glucose oxidase, and other chemicals that react with glucose.

• 3. Dipping the Strip: A small portion of the test strip is dipped into the urine sample. It is important to follow the manufacturer's instructions regarding the duration of immersion.

• 4. Reaction: If glucose is present in the urine, it reacts with the enzymes on the test strip. This reaction produces a color change on the strip.

• 5. Color Interpretation: After a specified reaction time, the color changes on the test strip are compared to a color chart provided by the test kit. The color chart indicates the concentration range of glucose in the urine.

• 6. Results: The results are interpreted as either "positive" or "negative." A positive result suggests the presence of glucose in the urine. This is an indication of hyperglycemia (high blood sugar levels). This could be associated with conditions such as diabetes. The presence of glucose in urine is called glucosuria or glycosuria.

Benedict's Test for Qualitative Test for Sugar in Urine

• Composition of reagent

  • · Cupric sulfate: 17.3 gm

  • · Sodium carbonate: 100 gm

  • · Sodium citrate: 173 gm

  • · Distilled water: 1.000 mL

• Principle: Copper sulfate reacts with sugar to form an insoluble cuprous oxide of red or yellow color.

• Procedure: Take 5 mL of Benedict's reagent in a test tube. Boil for a minute. Add 8 drops of urine, boil for 3 to 5 minutes, and cool it. Observe the color change

• Result

  • · Negative: No color change

  • · Trace: Pale green with cloudiness,

    • · 1+: Green

    • · 2+: Yellow

    • · 3+: orange to red

    • · 4+: Black red.

Fehling’s Test for Qualitative Test for Sugar in Urine

Fehling’s reagent consists of two solutions. Solution-A and Solution-B.

• Solution-A Composition

  • · Copper sulphate: 34.65 gm

  • · Distilled water: 500 mL.

• Solution-B

  • · Sodium hydroxide: 125 gm

  • · Sodium Potassium Tartrate: 173 gm

  • · Distilled water: 500 mL.

• Procedure: Take 2.5 mL of solution A and 2.5 mL of solution B in a test tube. Mix and boil. Add 8 drops of urine and boil again for 3 to 5 minutes. Fehling’s solution shows colour change as mentioned in the result of Bendict’s test.

Qualitative analysis of ketone bodies.

Normally, no or little amount of ketone is present in urine. Ketone bodies are the intermediate products of fat metabolism. Ketone bodies consist of acetoacetic acid, acetone, and Beta-hydroxybutyric acid. The presence of ketone bodies in urine is called ketoneuria. The formation of ketone bodies inside the human body is called ketosis or ketoacidosis. Ketosis occurs due to increased metabolism of fat in diabetes or a severe lack of carbohydrates in the diet.

• Causes of ketonuria

  • · Uncontrolled diabetes mellitus

  • · Chronic Starvation

  • · Pregnancy with vomiting

  • · Severe vomiting

  • · Glycogen storage disease.

Qualitative test for ketone bodies.

• Stick test

  • 1. Sample collection: Obtain a clean-catch or midstream sample of urine to minimize contamination.

  • 2. Test Strips: Test strips contain a pad or pads that react with ketones to produce a visible color change.

  • 3. Dipping the Strip: A small portion of the test strip is dipped into the urine sample. Ensure that the strip is fully immersed, and follow the manufacturer's instructions regarding the duration of immersion.

  • 4. Reaction: If ketones are present in the urine, they react with the reagents on the test strip. This reaction leads to a color change on the strip.

  • 5. Results: The results are interpreted as either "positive" or "negative." A positive result suggests the presence of ketones in the urine.

• Rothera’s test:

It is a very sensitive test for ketone bodies in urine. It is commonly used in most laboratories.

• Principle: Ketone bodies (Acetoacetic acid and acetone) with sodium nitroprusside develop a purple color complex in an alkaline environment.

• Method

  • 1. Add 5 mL urine is a test tube. Saturate it with ammonium sulfate. Add 0.5 mL 20 % aqueous solution of sodium nitroprusside or one crystal of sodium nitroprusside. Shake the tube and allow to stand for 10 minutes.

2. Carefully pour concentrated ammonia into a test tube. It forms a layer above the urine sample.

• Result: The formation of a purple color ring at the junction of urine and concentrated ammonia indicates ketone bodies (Acetoacetic acid and acetone) in the urine sample.

Bilirubin

Bilirubin is a blie pigment. Bilirubin attached to albumin is called conjugated bilirubin. Conjugated bilirubin is water soluble. Bilirubin is not normally present in urine. The appearance of bilirubin in urine shows liver dysfunction or obstruction of the bile ducts. Conditions such as hepatitis, cirrhosis, or gallstones lead to increased levels of bilirubin in the urine. The following methods are used to test bilirubin in urine.

• 1. Fouchet’s test for bilirubin in Urine

• Principle: Bilirubin is adsorbed on insoluble barium salts. Fouchit’s reagents is to oxidize bilirubin to a greenish-blue product.

• Reagents

  • · 10% barium chloride solution

  • · Fouchet’s reagent:

  • Trichloroacetic acid 25 gm

  • Distilled water 100 mL

  • 10% Ferric Chloride 10 mL

• Test

  • · Add 10 mL urine in a test tube then add 2-5 mL of 10% barium chloride solution. Mix and allow the mixture to stand for a few minutes to form a precipitate.

  • · Place the precipitate in a test tube on a piece of filter paper. Place this filter paper on a dry paper.

  • · Add Fouchet’s reagent on the precipitate.

• Result: A greenish-blue color indicates the presence of bilirubin in urine. The intensity of the green color shows the amount of bilirubin in urine.

• 2. Gamelin’s test:

  • Filter five mL of urine through filter paper. Allow filter paper to dry. Add a few drops of fuming nitric acid at the filter paper center. Colored rings will develop. Prominent green color rings indicate the presence of bilirubin in urine.

• 3. Reagent striped:

  • Commercial reagents strips are available to test bilirubin in urine.

Bile salt in Urine

• A trace of bile salt is present in the normal urine. Obstruction in the bile duct or drug-induced cholestasis leads to excretion of bile salt in urine. Hay’s Sulphur test is used to detect bile salts in the urine sample. Add 5 mL of urine to a test tube. Sprinkle a pinch of flower of sample on the surface of the urine sample in a test tube. Cool it below 15 degrees C.

• Result: Bile salt reduces surface tension. Sinking of the flower of sulfur indicates the presence of bile salt in urine

Urobilinogen in Urine

• Urobilinogen in urine becomes urobilin in a very short time. Thus, a fresh urine sample is used to detect Urobilinogen in urine. An increased level of Urobilinogen in urine indicates hemolytic jaundice. Normal adult urine of 24 hours contains 0.5 to 2.5 mg Urobilinogen.

Ehrlich’s qualitative test

It is used to detect Urobilinogen in urine.

• Principle: Ehrlich’s reagent reacts with Urobilinogen and porphobilinogen in urine to form aldehyde.

• Ehrlich’s reagent composition:

  • · Paradimethylaminobenzaldehyde 2 mL

  • · Concentrated Hcl 48 mL

  • · Distilled water 50 mL

• Method: Add 10 mL of freshly collected urine in a test tube. Add 1mL of Ehrlich’s reagent and mix well. Set aside for 5 minutes.

• Result Development of distinct red colour indicates presence of Urobilinogen in urine. Normal urine develop pink colour.

Blood in Urine

Under normal circumstances, there should be little to no red blood cells in urine. Red blood cells may present in urine during menstruation due to the migration of red blood cells from menstrual blood. The presence of blood in urine is called hematuria. Hematuria is due to various conditions such as urinary tract infections, kidney stones, trauma, or underlying kidney diseases. Hematuria can be microscopic (visible only under a microscope) or macroscopic (visible to the naked eye). The presence of hemoglobin without RBC in urine is called Haemoglobinuria.

Both red cells and hemoglobin show positive reactions to chemical tests. These qualitative analyses are conducted to detect hematuria and hemoglobinuria,

1. Benzidine test:

Reagent: Saturated solution of benzidine in glacial acetic acid. 2) Hydrogen peroxide.

Method:

• · Mix equal parts of reagent A and reagent B.

• · Add 4 mL urine to a test tube. Add 4 mL of mixed reagent

Result: The appearance blue color in the test tube indicates the presence of red cells or haemoglobin.

Detailed discussion on glycosuria and albuminuria

Glycosuria:

The presence of glucose in the urine is called Glycosuria. Under normal circumstances, the kidneys filter the blood to remove waste products. During this filtration glucose also enter into filtrate from kidney. Glucose is reabsorbed into the bloodstream. However, the kidneys may not be able to reabsorb all the glucose during high concentration of glucose in kidney filtrate. This leads to excrete glucose in urine. This occurs in diabetes mellitus or excess consumtion of sugar or carbohydrate food.

Causes:

• 1. Diabetes Mellitus: The most common cause of glycosuria is diabetes mellitus. In diabetes, the body either does not produce enough insulin (Type 1 diabetes) or does not use insulin effectively (Type 2 diabetes). This results in elevated blood glucose levels.

• 2. Other Conditions: Certain medical conditions, such as kidney disease, hormonal disorders, and liver disease, can also lead to glycosuria.

Symptoms: Increased thirst and urination, Fatigue, Unexplained weight loss, etc.

Diagnosis and Treatment:

• Diagnosis is typically confirmed through urine tests that detect glucose levels.

• Treatment involves managing the underlying cause, such as controlling blood glucose levels in diabetes.

Albuminuria:

The presence of albumin (a type of protein) in the urine is called Albuminuria. Normally, the kidneys filter the blood, preventing the passage of proteins into the urine. However, when the filtration process is damaged, albumin may leak into the urine.

Causes:

• · Diabetes Mellitus: Like glycosuria, diabetes is a common cause of albuminuria. High blood glucose levels damage the blood vessels in the kidneys. This leads to increased permeability and the leakage of albumin.

• . Hypertension (High Blood Pressure): Chronic high blood pressure also damages the kidneys and result in albuminuria.

• · Kidney Disease: Various kidney diseases, such as glomerulonephritis and nephrotic syndrome, can cause albuminuria.

• · Other Conditions: Certain systemic diseases and infections can contribute to albuminuria.

Symptoms

• · In the early stages, there may be no noticeable symptoms.

• · As the condition progresses, swelling (edema) in the hands, feet, and face may occur.

Diagnosis and Treatment:

• · Diagnosis is made through urine tests that measure the levels of albumin.

• · Treatment involves addressing the underlying cause, such as managing diabetes, controlling blood pressure, and treating kidney disease.

Clinical significance of urine analysis

Urine analysis (urinalysis) is a diagnostic tool used in clinical settings to evaluate and assess various aspects of a person's health. Urine analysis includes an examination of the composition of urine. Urine analysis provides valuable information about the body's metabolic processes, organ function, and potential health problems. The following are the clinical significance of urine analysis:

• 1. Detection of Medical Conditions:

  • Diabetes Mellitus: Urine analysis identifies elevated levels of glucose in the urine. It indicates poorly controlled diabetes.

  • Kidney Diseases: Abnormal levels of proteins (such as albumin) in the urine are signals of kidney dysfunction or damage.

  • Liver Diseases: Changes in the color of urine may be due to liver disorders. Such as darkening suspects bilirubin in urine that occurs in certain liver diseases such as jaundice.

• 2. Monitoring and Management of Chronic Diseases:

  • Diabetes Management: Regular urine analysis helps monitor blood sugar control in individuals with diabetes. It assesses the effectiveness of treatment.

  • Hypertension Management: Monitoring urine protein levels is essential in managing conditions like hypertension.

• 3. Assessment of Dehydration and Electrolyte Balance:

  • Specific Gravity: The concentration of urine indicates the body's hydration status. Low specific gravity may suggest dilute urine, while high specific gravity may indicate dehydration.

  • Electrolyte Levels: Abnormal levels of electrolytes in the urine indicate imbalances in the body's electrolyte homeostasis.

• 4. Identification of Infections:

  • Urinary Tract Infections (UTIs): The presence of white blood cells, red blood cells, or bacteria in the urine indicates a urinary tract infection.

  • Sexually Transmitted Infections (STIs): Infections such as gonorrhea or chlamydia are sometimes detected through urine analysis.

• 5. Screening for Drug Use:

  • Drug Metabolites: Some drugs and their metabolites are excreted in the urine. This allows the detection of drug use or abuse.

• 6. Pregnancy Monitoring:

  • Human Chorionic Gonadotropin (hCG): Urine analysis detects the presence of hCG. Hcg is a hormone produced during pregnancy. This is the confirmatory test of pregnancy.

• 7. Assessment of Renal Function:

  • Creatinine and Blood Urea Nitrogen (BUN): Levels of these substances in the urine state the renal function condition. They help to assess the kidneys' ability to eliminate waste products.

• 8. Evaluation of Metabolic Disorders:

  • Ketones: The presence of ketones in the urine indicates conditions like diabetic ketoacidosis or starvation.

• 9. Monitoring Treatment Response:

  • Chemotherapy and Medications: Urine analysis can be used to monitor the effects of certain medications or chemotherapy on the body.

Dr Pramila Singh