Determination of Urinary Protein
Determination of Urinary Protein

Determination of Urinary Protein

The urine analysis consist of three groups of tests:

  1. Physical examination of urine (Click the link to know about detailed physical examination of urine)

2. Chemical examination of urine

3. Biological examination of urine

This article have the thorough knowledge about the determination of urinary protein.

Chemical examination of urine is generally done by reagent strips which is commercially available. The chemical examination of urine helps to detect and/or measure various components, they are as below:

  1. Protein
  2. Glucose
  3. Ketone bodies
  4. Bile pigment
  5. Urobilinogen
  6. Bile salts
  7. Blood cells
  8. Nitrites
  9. Leukocyte esterase

Determination of urinary protein

Kidney excretes very less amount of protein in the urine (up to 150 mg/24 hours). These proteins include proteins from plasma (albumin) and proteins derived from urinary tract (Tamm-Horsfall protein, secretory IgA and proteins from tubular epithelial cells). This less amount of protein cannot be detected by routine tests. Regular determination of urinary proteins are necessary to maintain healthy lifestyle.

Clinical significance:

Proteinuria: when protein is found in urine in amount greater than 150 mg/24 hours in adults.

Causes of proteinuria: Glomerular proteinuria, Tubular proteinuria, Overflow proteinuria, Hemodynamic (functional) proteinuria and Post-renal proteinuria.

Tests for the determination of urinary proteins

Qualitative Test for determination of urinary protein

Qualitative Test for determination of urinary protein are as follows:

1. Sulfosalicylic acid (SSA) test

Principle: If proteins are present in urine, they are present in soluble form. However when we add sulfosalicylic acid reagent, these protein appears as white precipitate due to denaturation by the acidic reagent. Presence of turbidity in the urine mixture indicates the presence of proteins in urine.

Requirements:

  1. Test tubes
  2. Pasteur pipettes
  3. Sulfosalicylic acid (SSA)
  4. Urine sample

Procedure:

  1. Transfer 3-4 ml of centrifuged or filtered urine to a small test tube.
  2. Add 2-3 drops of SSA reagent on the top of specimen.
  3. Observe for the turbidity after 5 minutes.

Observation:

No.ObservationInterpretation
1.No turbidity formationProtein is absent
2.Formation of turbidityTrace
3.Formation of definite turbidity+
4.Turbidity + granulation++
5.Turbidity + granulation + flocculation+++
6.Turbidity + granulation + flocculation + solid precipitates or clumps++++
Result of SSA Test

2. Heat & Acetic acid test

Principle: Proteins get precipitate when boiled in an acidic medium and can be examine easily.

Requirements:

  1. Urine sample
  2. Test tubes
  3. Burner
  4. 10% Acetic acid solution

Procedure:

  1. Fill 2/3rd of test tube with urine, heat only the upper part of tube by keeping the lower part as control.
  2. Presence of phosphates, carbonates and proteins gives a white cloud formation in tube.
  3. Add 1-2 drops of 10% acetic acid solution in the tube, if the cloud persists it indicates the presence of protein (acetic acid dissolves the carbonates/phosphates).
  4. Cloudiness can be best see against black background.

Observation:

No.ObservationsInterpretation
1.No cloudinessNegative
2.Cloudiness appearsTrace
3.Cloudiness without granularity+
4.Granular Cloudiness++
5.Precipitation and flocculation+++
6.Thick solid precipitation++++

3. Heller’s test

Principle: Heller’s test is use for the determination of urinary proteins in urine. Heller’s test is a chemical test that shows that strong acids cause the denaturation of precipitated proteins. Concentrated nitric acid is added to a protein solution from the side of the test tube to form two layers. A white ring appears between the two layers if the test is positive.

Requirements:

Reagent

  1. Nitric acid (HNO3)
  2. Sample

Materials required

  1. Test tubes
  1. Test tube stand
  2. Pipettes

Procedure:

  1. Take 2 ml of concentrate nitric acid in a clean and dry test tube.
  2. To this, add 2 ml of urine or other sample. The sample should be poured from the sidewall of the test tube in an inclined position in order to form a layer of the sample above the nitric acid.
  3. The test tube is then observed for the formation of a white ring at the junction of the two layers.

Result and Interpretation:

  • Positive result: A positive result can form a white ring (precipitated protein) at the junction of the two distinct layers. This indicates the presence of proteins in the given sample.
  • Negative result: A negative result shows the absence of a white ring. This indicates the absence of protein in the sample.

4. Reagent strip test

A standard urine test strip may comprise up to 10 different chemical pads or reagents which react (change color) when immersed in, and then removed from, a urine sample. The test can often be read in as little as 60 to 120 seconds after dipping, although certain tests require longer.

The reagent area of strip is coated with indicator Bromophenol Blue and buffered to an acid pH 3. This area changes color in the presence of proteins.

For more details about reagent strip read following article.

Quantitative test for the determination of urinary proteins

Quantitative test for the determination of urinary proteins are as follows:

1. Esbach’s Albuminometer method

Principle: Esbach test is one of the oldest biochemical tests used for the determination of urinary protein like albumin when the urine is combined with citric and picric acid. The test is based on the principle of precipitation of albumin in the presence of organic acids. The organic acids (citric acid and picric acid) added to the sample exist as negative ions in the solution form. Similarly, the proteins in the urine sample exist as cations when the isoelectric pH of the protein is on the acidic side.

Thus, it is essential that the urine sample to be tested has to be acidic enough for the dissociation to take place. When the organic acids are added to the sample, the positively charged ions of proteins combine with the negatively charged ions of the organic acids. As a result, a salt of protein is formed with results in the formation of a precipitate. For the quantitative estimation of albumin, the precipitation formed in the tube is measured by the means of graduations present on the albuminometer tube used for the test. In order to estimate just albumin, a modified version of the Esbach test is developed. As per the modified method, urine is reacted with 10% potassium ferrocyanide, and acetic acid in a specially designed graduated conical flask. After about five minutes of centrifugation, it is said that every 0.1 ml of precipitate represents 1% albumin.

Requirements

Reagent

  • Esbach’s reagent: Add 10 grams of picric acid and 20 grams of citric acid to 1000 ml distilled water and mix well. A fresh reagent can give more accurate result.
  • Urine sample for determination of urinary protein

Materials required

  • Esbach’s albuminometer: Esbach’s albuminometer is a glass tube with markings. Two distinct markings can be seen where the mark ‘U’ indicates urine level and ‘R’ indicates the reagent level. From the base of the tube to the marking ‘U’ the tube is graduated from 0-12 gram. Each of these figures represents grams of albumin per 1000 ml of urine. These gradations help to measure the quantity of precipitate formed.
  • Pipettes
Esbach’s albuminometer

Procedure:

  1. Fill the Albuminometer with urine up to the mark”U”.
  2. The fill the Esbach reagent up to mark “R”.
  3.  Stopper the tube.
  4. Mix the contents well.
  5. Keep this tube in the standing position for 24 hrs.
  6. Look for precipitation and note the reading.
  7. Albumin is expressed in gms/L of urine.

Result and Interpretation:

  • Positive result: A positive result of the Esbach test shows the formation of yellow precipitate as soon as the reagent and urine sample comes in contact with one another.
  • Negative result: A negative result of the Esbach test shows the absence of yellow precipitate or the formation of precipitate after some time.

2. Biuret test:

Principle: This test is also known as Piotrowski’s test after the name of Gustaw Piotrowski, a polish physiologist, who document this test in 1857. Several other methods are: the modified Lowry test and BCA test. 

However, the mechanism of this test works through a series of principles. They are below. 

  • In the presence of alkaline, when Biuret reacts with dilute copper sulphate, a purple coloured substance is formed. The reason behind this colour is the formation of a chelate complex or copper coordination complex. 
  • Cu (II) or cupric ions create a chelate complex of violet colour, using oxygen of water and the unshared electron pairs of peptide nitrogen. 
  • Since this complex absorbs light in 540 nm, it appears violet. In the presence of protein, it changes its colour from blue to violet. 
  • Naturally, the colour intensifies as the number of peptide bonds increases in protein.  
  • Depending on this test’s principle, we can find the peptide bonds in any biological fluid. 
  • This reaction takes place in a compound having at least two, H₂N-CH₂-, H₂N-C, and H₂N-CS- or similar groups attached directly or via a nitrogen or carbon atom. 
  • One cupric ion is generally attach to six nearby peptide linkages through coordinate bonds.

Requirements:

  1. 1% alanine and 5% albumin or egg white (as positive control)
  2. Biuret reagents
  3. Deionised water (as negative control)
  4. Dry test tubes
  5. Water bath
  6. Pipettes 

Biuret Reagents

Biuret reagent is made of Copper sulfate (CuSO₄), sodium hydroxide (NaOH) and sodium potassium tartrate (also known as Rochelle salt). Despite the name, this reagent does not contain Biuret ((H₂N-CO-)2NH). It is a vital component of Biuret protein assay. 

Biuret Reagent Preparation

It is formed by mixing NaOH in a solution of CuSO₄, turning it alkaline. Following are the steps to yield 1000ml of Biuret reagent.

  • Take distilled water (500 ml) and dissolve pentavalent copper sulfate (1.5gm) and sodium potassium tartrate (6gm). 
  • Sodium potassium tartrate, a chelating substance that stabilises the copper ions. 
  • Now take 2 molar hydroxide (375 ml)
  • Now take a volumetric flask and mix two solutions. 
  • Finally, make it to 1000 ml by pouring distilled water. 

Procedure:

Using the following steps for determination of urinary proteins by biuret test, you can easily conduct this test. 

  1. First, take 3 dry and clean test tubes and label them as test, positive control and negative control.
  2. Now add 1 or 2 mL of the test solution (test), albumin (positive control) and deionised water (negative control) in the test tubes.
  3. Add Biuret reagent (1-2 mL) in each test tube. 
  4. Now shake the solution well and let it stand for 5 minutes. 
  5. Finally, observe how the colour changes. 
  6. For quantitative results, spectrophotometer can be used and optical density (O.D.) is taken against specific nanometre (nm).
  7. The graph is then plotted of O.D. versus absorbance.

Biuret Test Observation and Interpretations for Qualitative test:

ObservationInterpretation
No change in colourAbsence of proteins
Solution turns from blue to violetPresence of proteins
Solution turns from blue to pinkPresence of proteins

Biuret Test Observation and Interpretations for Quantitative test:

Graph of Absorbance versus Protein concentration

Precaution

Often ammonium and magnesium ions hinder this test. However, using excess alkali, it can be removed.

2 Comments

  1. Pingback: Urine Analysis - Physical Examination - Lifescience Stuff

  2. Pingback: Contributions of Louis Pasteur in Microbiology - Lifescience Stuff

Leave a Reply

Your email address will not be published. Required fields are marked *