REACTION OF NON PROTEIN NITROGENOUS SUBSTANCES (NPN)

 

REACTION OF NON PROTEIN NITROGENOUS SUBSTANCES (NPN)

 

                NPN Substances include all nitrogenous substances other than proteins. The NPN substances comprises Urea, Creatinine and Uric acid. Other nitrogenous substances of biological interest are ammonia, aminoacids, heme, purine nucleotides, pyrimidine nucleotides and small peptides. Urea creatinine and uric acid are included in this experiment.

 

I. Test for urea:

i.   Sodium hypobromite test:

ii.  Specific Urease test

 

II. Test for Creatinine:

i.   Jaffe’s test

 

III. Test for Uric acid:

i.   Phosphotungstic acid reduction test:,

 

I. TEST FOR UREA:

i) Sodium hypobromite Test:

                To 2ml of given solution add a few drops of freshly prepared sodium hypo-bromite solution. A brisk effervescence due to the evolution of gaseous nitrogen is observed.

 

ii) Urease Test:

                This test depends on the fact the enzyme urease hydrolyses urea into ammonia which makes the solution alkaline.

 

                To 2ml of given solution add a few drops of phenolphthalein indicator and a few drops of a freshly prepared urease solution wait for 15 minutes. Solution becomes pink due to the formation of ammonia indicating the presence of urea.

 

Test for creatinine:

Jaffe’s Test,

                Take 5ml of given solution in a test tube add 1ml of picric acid and make the mixture alkaline with NaOH solution (10%). Orange colour is due to formation of tautomer of creatinine picrate.

 

Test for Uric Acid:

Folin and Dennis Test (Phosphotungstic acid reduction test)

                To 2ml of given solution add 1ml of 10% Sodium carbonate and 1ml of phosphotungstic acid. Development of blue color indicates the presence or uric acid.