Urine Ketone Bodies And Bile Salts Analysis Pathology Notes

Urine Analysis

Urine Analysis Introduction

Urine analysis (urinalysis) reflects the state of function of the kidneys and urinary tract. It provides information about metabolic or systemic (nonrenal) disorders. Urine examination should precede all other invasive/noninvasive diagnostic investigations for renal function.

Examination of Urine:

Urine examination consists of:

1. Physical Examination,
2. Chemical Examination And
3. Microscopic Examination.

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Physical Examination

The volume of Physical Examination:

A healthy adult excretes about 600 to 2000 ml of urine in 24 hours. In infants, the volume is 300-600 mL/day.

Polyuria of Physical Examination:

Increased urine output (more than 2 liters in 24 hours).

Causes of Polyuria:

• Physiological: Increased fluid intake
• Pathological
• Diabetes mellitus (polydipsia—excessive intake of water)
• Chronic renal diseases
• Diuretic therapy
• Diabetes insipidus
• Primary aldosteronism.

Oliguria Physical Examination:

Decreased urinary output (less than 500 mL in 24 hours).

Causes of Oliguria:

• Restricted intake of fluid
• Excessive loss of fluid, for example In  Hemorrhage, burns, dehydration, and shock
• Renal diseases
  • Acute glomerulonephritis
  • Nephrotic syndrome
  • Acute tubular necrosis as in shock, burns, crush syndrome, incompatible blood transfusion, and heavy metal poisoning
• Addison’s disease.

Reaction (pH):

Normal urine is usually acidic with pH varying from 4.6 to 8. Urinary pH is measured as given below.

Litmus paper:

• Technique: The reaction of urine is determined with blue and red litmus paper. The urine when examined must be fresh as it turns alkaline on standing due to bacterial decomposition
  • Acidic urine turns blue litmus paper red
  • Alkaline urine turns red litmus paper blue
  • Both blue and red litmus papers turn reddish purple when the urine is faintly on the acid side (pH 7.0) or neutral.
• pH indicator paper strips/strip multistix method uses a methyl red and bromothymol blue double indicator system which can measure a pH from 5 to 9.

Specific Gravity:

It is used as a measure of the concentrating power of the kidney. The normal specific gravity of a 24-hour urine sample is 1.003 to 1.035, the average being 1.016. Specific gravity (SG) is measured by:

• Urinometer,
• Refractometer And,
• Dipstick Method.

The urinometer and refractometer methods are more accurate as compared to the dipstick method.

Urinometer Method:

• The urinometer is a specialized hydrometer.
• It consists of a weighted glass cylinder that flats in urine and has a calibrated stem to measure specific gravity at a given temperature.
• The scale in many urinometers is read in small divisions from 1.000 to 1060.
• It is usually calibrated in the range of 15° to 20°C (depending on the manufacturer).
• As the specific gravity varies with temperature, the reading obtained should be corrected to the room temperature by applying temperature correction.

Temperature correction: For every 3°C rise in room temperature beyond the calibrated temperature (15°C), add 0.001 to the recorded reading; and subtract 0.001 for every 3°C fall in temperature.

Technique Urinometer Method:

• The urine is poured into a wide-necked vessel (cylinder), and the urinometer is inflated, so that it does not touch the sides of the container.
• The specific gravity is read from the graduations which lie at a level with the true surface of the urine (lower meniscus).
• Observe the temperature of the urine. Check the temperature at which the urinometer is calibrated.

Refractometer Method:

It is based on the refractive index of urine and has the advantage of requiring only a few drops of urine.

Urinometer Metho:

SG is recorded by a change of color on the strip and compared with the color chart on the multistix bottle.

Causes of Urinometer Method:

• Increased specific gravity
  • Glycosuria
  • Dehydration: Restricted fluid intake, diarrhea, vomiting, fever, and excessive sweating.
• Decreased specific gravity
  • Excessive fluid intake
  • Diabetes insipidus
  • End-stage kidney
• Low and field-specific gravity: When the specific gravity is fixed at 1.010, this is known as isosthenuria. It is indicative of severe renal damage (chronic renal failure) with disturbance of both the concentrating and diluting abilities of the kidney.

Chemical Examination

Proteinuria

The presence of detectable protein in the urine is known as proteinuria. It indicates glomerular injury.

1. Heat and Acetic Acid Test

• Procedure:
  • Fill three-fourths of a test tube with clear urine.
  • Heat the upper part (1/3) of the urine (the lower part of the urine acts as a control for checking turbidity in the heated upper part).
  • Do not boil the urine, as it causes a mixing of the upper and lower parts of urine and traces of proteins cannot be appreciated.
  • The development of turbidity may be due to coagulation of proteins or due to phosphates.
  • Add a few (3–5) drops of 10% glacial acetic acid and if turbidity persists it is due to proteins (phosphates will dissolve).
• Interpretation: Depending on the amount of precipitate, results are interpreted as shown in Table.

2. Sulfosalicylic Acid Test:

Ths test detects all types of proteins (albumin, globulin, glycoproteins, and Bence Jones proteins).

• Procedure:
  • Take 2.5 mL of urine in a small test tube.
  • Slowly pour 2.5 mL of sulfosalicylic acid. Wait for 5 minutes.
• Interpretation: The presence of a cloudy precipitate indicates the presence of proteins in urine. However, it also precipitates mucus and Bence Jones proteins.

Interpretation of heat and acetic acid test:

3. Heller’s Test:

• Procedure: Take 5 mL of urine in a test tube and add a few drops of concentrated nitric acid.
• Interpretation: The presence of proteins is indicated by a white ring at the junction of urine and acid.

Dipstick Method of Heller’s Test:

Principle: The test is based on the protein error of pH indicators. At a constant pH, any color change in the indicator is due to protein.

• The reagent strip is impregnated with an indicator tetra bromophenol blue or tetrachlorophenol-tetra bromosulfophthalein buffered to pH 3.0.
• At this pH, it is yellow in the absence of protein.
• If the urine contains protein (which will elicit a pH change), it forms a complex with the indicator turning its color to green or bluish-green.

Interpretation: The strip is yellow in the absence of protein and variable shades of green develop depending on the type and concentration of protein present.

4. Quantitative Estimation of Proteins in Urine:

Protein excretion in a 24-hour urine sample is required in suspected cases of nephrotic syndrome (>3.5 g/24 hours) and orthostatic/postural proteinuria. This may be carried out by

Esbach’s albumino metermethod.

Procedure:

• Fill the luminometer with urine up to the mark U.
• Add Esbach’s reagent up to the mark R.
• Stopper the Esbach’s albuminometer, mix and allow it to stand for 24 hours.
• Take the reading from the level of precipitation in the albuminometer and divide the value by 10 to get the percentage of total proteins.

Precaution: The urine must be of acid reaction, clear, and should not be concentrated, in which case it must be diluted with water making allowance for the dilution in the fial calculation.

Causes of Proteinuria (albuminuria):

• Proteinuria may be classified as glomerular, tubular, lower urinary tract, prerenal and asymptomatic.
• It may also be classified depending on the amount of protein as heavy (>4 g/day), moderate (1 to 4 g/day), and mild (less than 1 g/day) proteinuria.

Reducing Substances In Urine:

Reducing substances are those compounds that reduce cupric ions (from copper sulfate in Benedict’s reagent) in an alkaline solution to cuprous ions (cuprous oxide). Such substances may be sugar or nonsugar.

Causes of proteinuria:

• Sugar: These include: glucose, fructose, pentose, galactose, lactose, maltose, and sucrose.
• Special tests are required to differentiate the sugar occurring in urine.
• Nonsugar: Ascorbic acid, uric acid, urates, glucuronides, chloroform, formaldehyde, salicylates, streptomycin, phenol, PAS, homogentisic acid (alkaptonuria), and creatinine.
  • Blood glucose level varies between 70 to 120 mg/dL. This may increase to 120 to 160 mg/ dL after a meal.
  • Normally, all the glucose in the blood is filtered through the glomerulus and reabsorbed at proximal tubules.
  • If the renal threshold (the lowest blood glucose level that will result in glycosuria) is exceeded (usually greater than 180 to 200 mg/dL), the excess glucose will not be reabsorbed into the blood and will be eliminated in the urine as in cases of diabetes mellitus.
  • The presence of detectable amounts of glucose in the urine is termed glycosuria.

Benedict Qualitative test (semiquantitative):

This test detects the presence of reducing substances in urine and is not specific for glucose.
Principle of Benedict test: The copper sulfate present in Benedict’s reagent reacts with the reducing substances in the urine which convert cupric sulfate to cuprous oxide in hot alkaline media.

Thus, this test is based on the reduction of cupric ions in Benedict’s solution to cuprous ions. In the absence of reducing substances in the urine, the color of the reagent remains blue.

The procedure of the Benedict Qualitative test: 

• Take 5 mL of Benedict’s (qualitative) reagent in a test tube.
• Boil to exclude the presence of reducing substance in the reagent.
• Add 8 drops (0.5 mL) of protein-free urine. Boil the mixture for 5 minutes and allow to cool.
• Note the color of the precipitate which is cuprous oxide formed due to the reduction of cupric sulfate of Benedict’s reagent.

Interpretation: The change of color from blue to green, yellow, and orange/red depends on the amount of sugar present.

Dipstick Method  of Benedict Qualitative Test:

Principle: Diastix/multi stix/dipstick contains:

• Glucose Oxidase
• Peroxidase
• Chromogen: O-Toluidine (Clinistix), Potassium Iodide (Multistix/Diastix).

Glucose present in the urine is oxidized by atmospheric oxygen in the presence of glucose oxidase to gluconic acid and hydrogen peroxide. The hydrogen peroxide, in the presence of peroxidase, oxidizes the reduced chromogen present in the dipstick to various shades of purple (oxidized chromogen)

The color change depends on the amount of glucose (semiquantitative) present in the urine.

The procedure of Glycosuria::

• The reagent area of the strip is completely immersed in fresh urine for 1–2 seconds. It is taken out and excess urine is drained off
• The change in color of the test area is compared with the color chart on the dipstick container exactly 30 seconds (or as per the manufacturer) after dipping the strip in the urine.

Cause of Glycosuria:

The substances that may give a positive reaction with Benedict’s test but negative with glucose oxidase strips are shown in Table.

Cause of Glycosuria:

Ketone Bodies

• The presence of ketone bodies in the urine is a measure of metabolic rather than renal function. Whenever there is a defect in carbohydrate metabolism, the fat is used as a source of energy.
• When increased quantities of fat are metabolized, there is increased production of ketone bodies which begin to accumulate in the blood and are subsequently excreted in the urine.

Ketone bodies are three metabolically related compounds:

• Acetoacetic (diacetic) acid
• β-hydroxybutyric acid
• Acetone.

If urine is left at room temperature, acetoacetic acid slowly converts into acetone.

Causes of false positivity by nonsugar-reducing substances:

1. Rothera’s Test:

The procedure of Rothera’s Test:

• Take 4 mL of urine in a test tube.
• Add a few crystals of sodium nitroprusside and saturate the urine with ammonium sulfate by mixing vigorously.
• Overlay with a few drops of liquor ammonia along the wall of the tube.

Interpretation: The development of a purple ring indicates the presence of acetoacetic acid/acetone or both. A brown or red color is of no significance.

2. Gerhardt’s Test (ferric chloride test):

This is neither a very specific nor a sensitive test. The test detects acetoacetic acid.

The procedure of Gerhardt’s Test :

• Take 8 mL of urine in a test tube.
• Add 10% ferric chloride solution drop by drop; a precipitate of ferric phosphate forms.
• Add more ferric chloride solution till the ferric phosphate precipitate disappears.
• Filter it and add an excess of ferric chloride to the filtrate.

Interpretation: A red color of the filtrate indicates a positive test.

Dipstick Method of Gerhardt’s Test :

Principle: Dipstick contains buffers and sodium nitroferricyanide, which react with acetoacetic acid in the urine to form a pink-maroon color in 15 seconds. The strips detect acetoacetic acid and not acetone. However, acetone can be detected if glycine is incorporated into it.

Causes of ketonuria:

• Diabetic ketoacidosis
• Starvation
• Prolonged vomiting or diarrhea
• Prolonged febrile illness
• Von Gierke’s disease
• Eclampsia

Bilirubin (Bile Pigment)

• Tests for bilirubin in urine provide information concerning metabolic or systemic disorders, especially liver function. Bilirubin is a breakdown product of hemoglobin and is normally not present in urine.
• Even trace amounts are clinically significant and only conjugated bilirubin is found in urine.
• Bilirubinuria causes yellow-brown to greenish-brown urine and forms yellow foam on shaking. Bilirubin is found only in freshly voided urine which upon standing is oxidized to biliverdin.

Fouchet’s Test:

• Procedure:
  • Take 10 mL of urine in a test tube and add 3 mL of 10% barium chloride solution.
  • Mix the two and filter the mixture through a filter paper. Bilirubin along with barium salt remains on fitter paper.
  • Add a few drops of Fouchet’s reagent onto the fitter paper.
• Interpretation: Green or blue color indicates bilirubinuria.

Note: The foam test is a simple test to detect bile pigments at the bedside. Take urine in a test tube and shake the urine. If bile pigments are present in the urine, the foam on top will be yellow in color.

Dipstick Method of Fouchet’s Test:

Principle: The test is based on a diazo reaction. Bilirubin in the urine couples with a diazotized dichloroaniline (the content of strip) in a strongly acidic medium to form a colored compound namely azobilirubin.

Causes of Bilirubinuria:

• Obstructive jaundice: Urine shows bilirubin without urobilinogen
• Hepatocellular jaundice: Bilirubin is absent in the urine of patients with hemolytic jaundice.

Urobilinogen

Urobilinogen is normally present in urine in trace amounts (1–2 mg/dL) and is insufficient to cause a significant positive reaction. Whenever, the liver is unable to efficiently remove the reabsorbed urobilinogen from the portal circulation (e.g. liver diseases, hemolytic anemia) more urobilinogen than normal is routed through the kidney and hence excreted in the urine.

Ehrlich’s Test:

• Procedure:
  • Take 2.5 mL of urine in a test tube.
  • Add 2.5 mL of Ehrlich’s aldehyde reagent (para dimethyl amino benzaldehyde). Mix well by inversion.
  • Add 10 mL of saturated sodium acetate solution and mix.
• Interpretation: Pink to cherry red color indicates the presence of urobilinogen.

Dipstick Method Ehrlich’s Test:

Principle: This method is based on Ehrlich’s test. The strip is coated with p-amino benzaldehyde with a color enhancer. It reacts with urobilinogen in a strongly acidic medium to produce a pink-red-colored compound. The color is matched with the color chart on the container of the dipsticks.

Causes of Increased Urobilinogen in Urine:

• Hemolytic anemias (without bilirubin in urine)
  • Thalassemia
  • Sickle cell anemia
  • Hereditary spherocytosis
• Liver diseases
  • Preicteric phase of infective hepatitis
  • Drugs or toxic hepatitis
  • Cirrhosis.

Causes of Decreased/Absent Urobilinogen in Urine:

In obstructive jaundice, bilirubin does not reach the intestine and hence is not converted into urobilinogen.

Bile Salts

Bile salts are composed of a mixture of bile acids and glycine or taurine. Two important bile salts are sodium and potassium salts of glycocholates and taurocholates. Normally, bile salts are not present in urine.

Hay’s Sulfur Test:

The procedure of Hay’s Sulfur Test:

• Take 10 mL of urine in a wide-bore test tube (2 cm diameter or more) or a small beaker.
• Sprinkle sulfur powder over its surface, and watch for 5 minutes.

Interpretation: Sulfur powder sinks to the bottom of the test tube in the presence of bile salts in urine.

Precaution: Since soap can give false positive results, the test tube should be clean.

Causes: Hepatocellular and obstructive jaundice.

Tests For Blood In Urine

These tests detect hematuria, hemoglobinuria, or myoglobinuria.

• Hematuria: Presence of red cells in urine, e.g. renal stones, renal cell carcinoma.
• Hemoglobinuria: Presence of free hemoglobin in the urine, for example, Intravascular hemolysis.
• Myoglobinuria: Presence of myoglobin in urine, for example, Crush injury to muscle, strenuous exercise.

1. Benzidine Test:

Procedure of Benzidine Test:

• Dissolve a small amount (knife-point full) of benzidine in 2 mL of glacial acetic acid and add an equal volume of 3% hydrogen peroxide.
• From the above, take 2 mL in another test tube add 2 mL of previously boiled and cooled urine, and mix.

Control for the test: If the test is negative, add a drop of blood to the above test tube. It should give a blue color. This is to confirm the potency of reagents, especially hydrogen peroxide, and excludes a false negative reaction.

Interpretation: The appearance of blue color indicates the presence of blood.

 Precaution: The presence of hypochlorite (bleach) and microbial peroxidase can cause false positive results. Benzidine is carcinogenic.

2. Orthotoluidine Test:

The procedure of the Orthotoluidine Test:

• Take 2 mL of urine in a test tube.
• Add 1 mL of orthotoluidine solution in glacial acetic acid.
• Add a few drops of hydrogen peroxide.

Interpretation: The appearance of blue or green color indicates the presence of blood.

Dipstick Method of Orthotoluidine Test:

Very small amounts of hemoglobin/RBCs are detected by the dipstick method.

Principle:

• The test is based on the liberation of oxygen from peroxide in the reagent strip by the peroxidase-like activity of heme present in hemoglobin.
• The liberated oxygen changes the color of the chromogen. The reagent area is impregnated with organic peroxide and the chromogen is ortho toluidine/tetramethylbenzidine.

Causes of Hematuria:

Urine is red-colored in severe hematuria and smoky in mild hematuria. RBCs are demonstrable in urinary sediment. The causes of hematuria are shown in Table.

Causes of hematuria:

Multistix Reagent Strips for Urine Testing

• These have single/multiple discrete cellulose squares which are impregnated with reagents for testing glucose, protein, pH, specific gravity, hemoglobin, ketone bodies, bilirubin, and urobilinogen.
• There are single test strips, for example, Diastix for glucose and multistix for multiple tests.

Automated Urinalysis

Fully automated urine analyzers provided with automatic functions are presently available, for example, Uriplus.