Urinalysis : Chemical Examination

Chemical Examination of Urine

The routine analysis of urine includes chemical test for protein, glucose, ketone bodies, occult blood, bile salts, bile pigments and urobilinogen. 

Proteins in urine

Urine normally contains only a scant amount of protein which derives both from blood and urinary tract itself. Mainly albumin is filtered from nephrons due to low molecular weight others are reabsorbed by renal tubules. Other protein includes serum or plasma globulin, mucus or mucin, hemoglobin, bence jones protein.

Determination of protein in urine

Principle

All the methods are based on the principle of precipitation of protein by chemical agents or coagulation by heat.

Methods

Qualitative tests

• If urine is alkaline make it slightly acidic by adding 3% glacial acetic acid.
• Turbid urine should be filtered or centrifuged and supernatant should be used

• Heat and acetic acid test
• Sulphosalicyclic acid test
• Purdy’s modification 

Quantitative test for albumin

Qualitative test and semi quantitative test have limitation that they can’t detect the exact amount of protein excretion. So quantitative test is done on 24 hr urine

Mainly 2 methods are used for this purpose which uses picric acid for precipitation in different proportion methods are: 

• Esbach’s method
• Aufrecht’s method

The most commonly used test are-

A.     Heat and Acetic Acid Test 

1. Place 5 to 10 ml of clear urine in test tube
2. Boil the upper portion over a flame.
3. If turbidity develops add 1-2 drops of glacial acetic acid. Sometimes turbidity may be due to phosphate or carbonate precipitation. it is so then glacial acetic acid clear up the turbidity .if it is due to protein then precipitation will be there after the addition of acetic acid
4. Reboil the specimen
5. If turbidity is present protein is present .if there is no turbidity at upper portion then protein is absent.
6. Grade the turbidity as follows:

• Negative : No cloudiness
• Trace: Barely visible cloudiness.
• 1+ : definite cloud without granular flocculation
• 2+ : heavy and granular cloud without granular flocculation
• 3+ : densed cloud with marked flocculation.
• 4+ : thick curdy precipitation and coagulation

Sulphosalicyaclic Acid Test

Reagents

3% sulphosalicylic acid is prepared

Sulphosalicyaclic acid         3.0 gm

Distilled water               100 ml

Procedure

1. Place 3-4ml of clean urine in test tube
2. From the side of tube add 2-3 drops of sulphosalicylic acid on top.
3. Let it stand for 5 minutes.
4. Observe the turbidity.

Result

• No formation of turbidity at upper portion of urine indicates absence of protein
• Formation of turbidity indicates presence of protein.

Turbidity is graded as follows:

• Trace  cloudiness against dark background
• 1+       dense cloudiness.
• 2+       cloudiness with granules and definite flocculation.
• 3+       cloudiness with flocculation.
• 4+       cloudiness with precipitation.

Clinical Significance

Proteinuria can occur mainly due to

1. Glomerular damage

2. Defect in reabsorption of process of tubules

Always proteinuria in not pathological. So there are mainly 3 types of proteinuria.

A. Accidental Proteinuria

Due to contamination of urine with vaginal seminal discharge after prostatic massage and derivation from diseased condition of genital tract or bladder accidental proteinuria is seen.

B. Functional Proteinuria

Non pathological proteinuria also called physiological albuminuria mainly seen in strenuous exercise, phrexia, exposure to cold, congestive heart failure hypertension atherosclerosis pregnancy dehydration fever if person stand in upright position for longer period. (Postural or orthostatic proteinuria)

C. Renal Proteinuria

Any condition resulting in increased permeability of urinary tract surfaces or in transduction such as glomerulonephritis diabetes nephritis associated with SLE, pyelonephritis, hereditary fructose intolerance, cystitis urinary tract, malignancies, heavy metal poisoning, eclampsia, amyloidosis, sarcoidosis, sickle cell disease, renal transplant rejection, multiple myeloma, degenerative and irritative condition and lower urinary tract.

Note

• Orthostatic proteinuria can be differentiated from pathological proteinuria by testing 2 urine samples ,one collected immediately after rising and one collected after patient has been in upright position for 3 hour or longer.
• During heat and acetic acid

1. If cloudiness is seen it may be due to phosphate or carbonates confirm by adding 3% glacial acetic acid. if it is due to protein cloudiness persist and if it is due to the phosphate the cloudiness disappears and if it is due to carbonate cloudiness disappears with effervescence.
2. If cloudiness is disappeared when nitric acid is added then it is due to mucin and nucleoprotein. 

If cloudiness appears with the tube is being heated but disappears when boiling point is reached bence jones protein is present. Bence jones protein is low molecular weight protein which is easily filtered through the glomerulus. It has unusual solubility .it precipitates when heated at 40-60 degree centigrade and becomes soluble when boiled and reappears on cooling. it is seen in urine in multiple myeloma where there is malignant proliferative of plasma cells in bone marrow.

• Heat and acetic acid test detect as little as 2-3 mg/100 ml of protein.
• Sulfosalicylic acid detects 5 mg/100 ml of protein.

Sugar in Urine

Normally glucose is virtually absent from urine .the renal threshold for glucose ranges from 160-200mgm/dl depending on the individual .that is blood sugar must rise to its renal threshold before glucose will appear in urine. When glucose is present in urine it is called glysosuria .other less important sugar that may appear in urine are lactose galactose pentose which may give false positive results for glucose. So specific test must be performed for differentiating glucose from other sugars present in urine.

Methods.

1. Determination of Glucose.

• Benedicts test
• Fehlings test

2. Determination of Lactose

• Yeast fermentation test
• Osazone test.
• Rubners test.

3. Seliwanoffs Test for Fructose

4.  Bial’s Test For Pentoses

Determination of Glucose by Benedicts Test

Fig. Benedict's test (Negative, 1+, 2+, 3+ and 4+ from left to right)

Principle

When benedicts qualitative reagent is heated with 8 drops of urine glucose present in urine reduces cupric ions present in reagent to cuprous ions. Alkaline medium is provided to the reaction by sodium carbonate present in reagent .the color changes to green yellow orange or red according to concentration of glucose in urine.

Reagent

Benedict’s qualitative reagent preparation:

Sodium citrate 1.73 gm.

Sodium bicarbonate 100 gm.

• Place in about 900 ml of distilled water.
• Boil for 2-3 minutes and add 17.3 gm of cupric sulfate
• Make final volume up to 1 liter
• The reagent is stable at room temperature.

Procedure

• Pipette ml of benedicts reagent in test tube
• By using Pasteur pipette add 8 drops of urine
• Heat carefully or place in boiling water bath for 5-10 mins
• Cool under tap water.

Result

• No change in color i.e. blue: Absence of sugar.
• Pale green with slightly cloudy: Trace
• Definite cloudy green: 1+
• Yellow to orange precipitate with supernatant fluid pale blue: 2+
• Orange to red precipitate supernatant fluid pale blue: 3+
• Brick red precipitate supernatant decolorized: 4+

Clinical significance

In general glucose is seen in urine in 2 conditions

A. When blood sugar is elevated

B. When blood sugar is not elevated but renal tubular absorption-glucose is impaired.

Glucose in urine is mainly seen in diabetes mellitus.

It is increased in

1. Any cause of increased blood glucose.
2. Rapid intestinal absorption (post gastrectomy dumping normal pregnancy)
3. Endocrine disorders other than diabetes milletus like thyrotoxicosis, gigantism.acromegaly, Cushing syndrome.
4. Major trauma stroke myocardial infarction or circulatory collapse cerebral hemorrhage
5. Burns oral steroid therapy infection pheochromocytoma
6. Glycogen storage disease, obesity, sepsis, carcinoma of pancrease, fanconi’s syndrome, cystinosis.

Note

1. If benedicts show more than 2.5% sugar urine should be diluted.
2. If benedicts test is positive then it is necessary to confirm it by using glucose oxidase uristix
3. Sugar in urine is also detected in gestational diabetes oral glucose tolerance test spot test during post prandial blood glucose.
4. Benedict’s reagent gives false positive in certain non-carbohydrate also such as uric acid creatinine salicyaclic acid homogentisic acid and melanogen.

Fig. Multiple Uristix

Ketone bodies

The term ketones refer to 3 intermediate product of fat metabolism, they are acetone acetoacitic acid and buta hydrooxybutyric acid.

Ketone is found when there is excessive fat metabolism .excessive fat metabolism occurs in various situation

• Impaired ability to metabolize carbohydrate
• Inadequate carbohydrate intake
• Excessive carbohydrate loss
• Increased metabolic demand.

Method

1. Rothera's test for acetone.

2. Gerhard's test for diacetic acid

3. Lindeman's test for diacetic acid

4. Han’s method for betahydroxybutyric acid.

5. Tablet test

Principle

Nitroprusside used in this test reacts with both acetone and acetoacetic acid in presence of alkali (NH₄OH) to produce permanganent calomel red ring at the junction

Requirements

1. Test tubes
2. Rothera powder mixture

• Sodium nitroprusside
• Ammonium sulphate
• Liquior ammonia solution

Procedure

1. Transfer about  5 ml of urine to a test tube
2. Saturate with ammonium sulphate
3. add 1 crystal of sodium nitroprusside
4. Layer the liquor NH₄OH  on the side of the tube
5. Observe permanganate calomel ring at the junction of two layers.

Clinical Significance

Increased In

• Diabetes mellitus
• Propanol poisoning
• Severe starvation.
• Severe carbohydrate restriction
• Anorexia
• Fasting
• Fever
• Prolonged vomiting
• Lactic acidosis
• Salicyclate toxicity.

Note

1. In diabetes mellitus impaired ability to metabolize carbohydrate takes place. as carbohydrate cannot be used to meet the body energy need, fats are burned which leads to the presence of ketones in the urine.
2. Acetoacetic acid oxidizes rapidly to form acetone therefore test must be carried out in fresh urine specimen.
3. Individuals receiving levadopa paraldehyde pyridium and phathalein compound may produce false positive result when tested for ketonuria. Presence of salicylates give false negative result.
4. When sugar is found in urine, the urine should be tested for ketone.

Occult Blood

The term occult means hidden. Blood may be present in the urine as either red blood cells or hemoglobin. If enough blood is present the color of sample may be range from pink tinged to red to brownish black.

Tests

1. Microscopical Examination

2. Chemical Examination

• Benzidine test
• Guaiacum test
• Gregersens test
• Ortho-toluidine test.

3. Spectroscopic Test

Benzidine Test

Principle

The peroxidase activity of hemoglobin present in urine decomposes hydrogen peroxide and the liberated oxygen oxidized benzidine to form a green- blue colored complex.

Procedure

1. Place a  pinch of benzidine in a test tube
2. Add 2- 3 drops of 5% glacial acetic acid.
3. Mix well
4. Add 2 ml of hydrogen peroxide solution.
5. Transfer supernatant to a test tube label as T
6. Add few drops of urine and observe the color.

Clinical significance

1. Hematuria

Presence of more number of red blood cells in urine is called hematuria which is associated with disease of or damage to the genitourinary tract .other disorder commonly used associated with hematuria includes acute infection chronic glomerulonephritis tuberculosis of kidney nephritic syndrome toxic damage to glomerulus malignant hypertension infarction renal calculi trauma to kidney, acute cystitis, calculi, tumors in the ureter or bladder and kidney stones. In other clinical conditions such as bleeding disorder (leukemia, thrombocytopenia, coagulation factor deficiency, sickle disease or traits, scurvy), use of anticoagulant drugs.

2. Hemoglobinuria

It is the presence of free hemoglobin in urine as a result of intravascular hemolysis.

Causes of hemoglobinuria

Acute

• Incompatible blood transfusion
• Hemolytic anemia due to drugs and chemicals.
• Favism.
• Paroxysmal cold hemoglobunuria.
• March (exertional) hemoglobunuria.
• Hemolytic anemia associated with eclampsia
• Hemolytic uraemic syndrome.
• Hemolytic anemia due to burns
• Snake and spider bites.

Chronic

• PNH
• Cardiac hemolytic anemia
• Cold haemagglutination disease.

3. Myoglobinuria

Myoglobin is the haem protein of striated muscle. Myoglobin is very toxic to the renal tubules and in large amounts it is associated with acute renal failure.

Clinical Conditions

1. Myocardial infarction
2. Infarction of large skeletal muscle
3. Destruction of muscle with crush injury heat stroke electric shock
4. Trauma

Note

1. False positive result is seen in women during menstruation due to contamination of urine with menstrual blood. So this test should be avoided during menstruation cycle.
2. Free Hb is not normally found in the urine .instead any Hb that could be presented to the glomerulus combines with heptoglobin. The resultant Hb  heptoglobin complex is too large to pass through the glomerular membrane .If the amount of free Hb exceeds the amount of heptoglobulin , however the Hb will pass through the glomerulus and ultimately be excreted into the urine .Any disorder associated with hemolysis of red blood cells and resultant release of Hb may lead to the appearance of Hb in urine
3. Hematuria can be differentiated from hemoglobunuria by doing microscopical examination. In hematuria RBC seen in microscopy. In hemoglobunuria, RBC cannot see even though the test for occult blood is positive.
4. This test can be done for stool as occult blood for stool.

Bile in Urine

Bilirubin, bile salt, bile pigment, urobilin, urobilinogen are the constituents of bile.

Determination of Bile Salt

Hay's test

Principle

Bile salts when present lower the surface tension of urine. When sulphur powder is added to the urine, sulphur particles sink   to the bottom of the tube. In the case of normal urine, it will float on the surface.

Procedure

1. Place about 10 ml of urine in a test tube
2. Sprinkle a little dry sulphur powder on to the surface of urine.
3. Observe sulphur particles

Methods

1. Foam test
2. Gmelin's test
3. Smiths test
4. Fouchet's test
5. Ehrlich's aldehyde test
6. Schlesingers test

Foam test

Shake some urine in a test tube. If the foam on the top is yellow, the bile pigments are present.

Fouchet’s test

Principle

When barium chloride is added to urine it combines with sulphate radicals in urine and precipitate of barium phosphate is formed. If bile pigments are present in urine, they will adhere to these large molecules. Ferric chloride present in fouchet reagent then oxidizes yellow bilirubin in presence of trichloroacetic acid to green bilverdin.

Requirements

• Test tube
• Pasteur pipette
• Fouchet’s reagent
• Filter paper

Fouchet’s reagent:

Trichloroacetic acid     25 gm

Distilled water     100 ml

10 % ferric chloride solution   10 ml

Procedure

1. 10 ml of urine + 2.5 ml of barium chloride
2. Filter
3. Unfold the filter paper and spread it on the dry filter paper.
4. Allow 1 drop of Fauchet’s reagent on the precipitate
5. A green or blue color indicates presence of bilirubin.

Ehrlichs Aldehyde Test for Urobilinogen

Procedure

1. Take 5 ml of urine in test tube and add half volume i.e. about 2.5 ml of barium chloride.
2. Mix well and filter.
3. Take 2.3 ml of filtrate and add 0.5 ml of aldehyde reagent.
4. Allow to stand for 3 mins.
5. View the top column of urine against a white background.
6. A pink color denotes the presence of urobilinogen.
7. Repeat the test with 1:10, 1:20, 1:50, 1:100, 1:200 dilution and report a terms of highest dilution giving a positive reaction.

Ehrlich's reagents

Paradimethylaminobenzaldehyde    2 gms.

20% HCl                                   100 ml.

Schlesingers Test for Urobilin

Procedure

1. Take 10 ml of urine and 6 drops of tincture of iodine in a test tube.
2. Take 1 gm of powdered zinc acetate and 10 ml 95% alcohol in another test tube.
3. Mix by pouring a into b and vice versa repeatedly until the solid zinc acetate has mostly gone into solution.
4. Filter.
5. Examine the filtrate.
6. A green is due to compound of zinc with urobilin, confirm spectroscopically absorption band junction of green and blue.

Clinical significance

1. Determination of bile salts, bile pigments, and urobilinogen is useful in the diagnosis of jaundice.
2. Bilirubin may be found in urine in liver disease and is usually found in clients who have biliary tract obstructions.
3. Conjugated bilirubin appearing in urine generally indicates that there is excess conjugated bilirubin in blood stream.
4. Bilirubinuria is seen when intracanalicular pressure rises secondary to periportal inflammation, fibrosis or hepatocyte swelling.
5. Gallstones in the common bile duct or carcinoma of the head of pancreas are possible sources of extra hepatic biliary obstruction leading to bilirubinuria.
6. Congenital hyperbilirubinemia seen in gilberts disease or crigler najjar disease.
7. When liver cells are damaged, excreation of urobilinogen in the bile decreased, where as its urinary excreation is increased. This may be seen in cirrhosis, hepatitis and congenital heart failure with congestion of the liver.
8. Excessive urobilinogen also may be found in the urine of those with liver disease or hemolytic disorder.

Note:

Condition	Urine bilirubin	Urine urobilinogen
Bile duct obstruction	+ + +	negative
Liver damage	+ or -	+ +
Hemolytic disease	negative	+ + +

• The bilirubin in urine is made confirmed by doing confirmatory bilirubin test called as diazo test.
• The Fouchet’s test is also called Harrison spot test.
• Fresh urine sample should be used for bilirubin determination because exposure of urine to light and room air may give false negative result .large amount of ascorbic acid and nitrates also give false negative result.

Acidic urine will result in decreased urinary level of urobilinogen. High levels of nitrates in the urine also may cause false negative results in test for urobilinogen.