CHEMICAL EXAMINATION


Chemical constituents of a normal urine sample are shown in Table 50.5.

Proteinuria

Normal urine may contain up to 150 mg of protein/day. The average urine protein concentration ranges from 2–10 mg/dL and is not detectable by routine test. 

The presence of detectable protein in the urine is known as proteinuria. It indicates glomerular injury. If turbid, filter or centrifuge the urine before testing.


Tests for protein

a. Heat and acetic acid test

b. Sulfosalicylic acid test

c. Heller’s test

d. Dipstick method

(A). Heat and Acetic Acid Test

Principle

Heat induced coagulation of proteins and precipitation. 
Coagulation can be further enhanced when 
drops of acetic acid are added.

Fig. 50.2: Method of performing heat and acetic acid test


•• Procedure (Fig. 50.2): Fill three-fourth 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 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 50.6.

•• Precaution: 

Before testing for proteinuria, the pH of the urine should be checked. If alkaline, the urine should be acidified with a few drops of glacial acetic acid.

(B). Sulfosalicylic Acid Test


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

Principle

Cold precipitation of proteins by a strong acid. 
More sensitive and reliable than heat and acetic acid test.


•• Procedure
–– Take 2.5 mL of urine in a small test tube

–– Slowly pour 2.5 mL of 3% sulfosalicylic acid. Wait for 10 minutes.

•• Interpretation (Table 50.7):

Presence of a cloudy precipitate indicates the presence of proteins in urine. However, it also precipitates mucus and Bence Jones proteins.

•• Precaution:

Centrifuge/filter the urine if turbid and use clear supernatant because turbidity interferes with the final reading.


(C). Heller’s Test

Principle

Cold precipitation of proteins by a strong acid.

•• Procedure: 

Take 5 mL of urine in a test tube and add a few drops of concentrated nitric acid.

•• Interpretation: 

Presence of proteins is indicated by a white ring at the junction of urine and acid.

•• Precaution:

Filter urine if turbid as it interferes with the final reading.

(D). Dipstick Method

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-tetrabromo-sulfophthale in buffered to pH 3.0. At this pH it is yellow in the absence of protein.

If urine contains protein (which will elicit a pH change), it forms a complex with the indicator turning its color to green or bluish green.

•• Procedure

–– 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 by gently tapping the edge of the strip against the side of the urine container.

–– Change in the color of the strip is compared with the reference color chart for proteins on the dipstick container. Strips are sensitive and able to detect as low as 20 mg/dL of albumin in the urine.

•• 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.

Fig. 50.3: Esbach’s albuminometer used for quantitative measurement of proteins in urine


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 albuminometer (Fig. 50.3) method.

Principle

Cold precipitation of proteins by a strong acid.

•• Procedure

–– Fill the albuminometer 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 final calculation.

Causes of Proteinuria (Albuminuria) (Box 50.2)

Categories of proteinuria

•• Proteinuria quantification:

 Classified depending on the amount of protein in the urine as heavy (>4 g/day), moderate (1–4 g/day) and mild (less than 1 g/day) proteinuria.

•• Qualitative categories of proteinuria:

 Classified depending on the structure involved as 

(1)renal (glomerular causes/pattern, tubular cause/pattern), 

(2) prerenal, and 

(3) postrenal.

•• Postural (orthostatic) albuminuria: 

In this condition, protein is seen in the urine only when the patient is active on his feet. At night when recumbent, the early morning sampleis negative for protein. It may be related to renal congestion and ischemia when patient is active.

•• Bence Jones proteins:

Bence Jones proteins are light chains of immunoglobulins, secreted in multiple myeloma. It may also be found in macroglobulinemia and lymphoma. Bence Jones proteins precipitate at temperature between 40°C and 60°C, and redissolve near 100°C. It reappears on cooling to 40–60°C.

•• Acetic Acid Test for Bence Jones Protein:

–– Procedure

◆◆ Take 5 mL of clear urine in a test tube and 1 mL of acetate buffer.

◆◆ Heat the urine in a water-bath with a thermometer inside.

◆◆ At 40°C, Bence Jones proteins start precipitating till 58°C when the precipitation is complete.

◆◆ If urine is heated beyond 60°C, Bence Jones proteins dissolve in the urine and reappear on cooling at 60°C.

However, the best method for detection of Bence Jones protein in urine is by protein electrophoresis.

•• Combined albuminuria with Bence Jones proteinuria:

In cases where both albumin and Bence Jones proteins are present, boil the urine and filter. Precipitated albumin will be filtered out. Take the filtrate and heat it to 40–60°C when Bence Jones proteins precipitate. Bence Jones proteins dissolve on boiling.

•• Microalbuminuria:

Screening tests for urine protein are not sensitive enough to detect very small quantities of protein. Microalbuminuria is the presence of albumin in urine above the normal level but below the detectable range of conventional methods. It is defined as the persistent elevation of the urinary albumin excretion of 20–200 mg/L (or 20–200 mg/min) in an early morning urine sample. It indicates early and possibly reversible glomerular damage.