URINARY SCREENING
TEST FOR INBORN
ERRORS OF METABOLISM
.
Inborn errors of metabolism(IEM) are metabolic disorders due to deficiency or
absent of an enzyme in a metabolic pathway. This results in following.
• Accumulation of substrate
• Decrease synthesis or absent of product
• Division of substrate in to alternative pathways producing undesirable
compounds
Ferric chloride test
A) Procedure: to 1 ml of urine sample add 2 drops of 10% ferric chloride solution. Mix gently
observe the colour change .
Observation: The positive result will gives a transient blue green colour.(it is due to presence
of phenyl pyruvate).
Inference : PHENYLKETONURIA
• It is due to the deficiency of phenylalanine hydroxylase.
B) Observation : Positive result will gives blue colour.
Inference : ALKAPTONURIA
• This is based on the observation that the urine becomes black on standing when it become
alkaline.
• The metabolic defect is deficiency of homogentisate oxidase.
Dinitro phenyl hydrazine test (DNPH)
A)Procedure: To 2ml of urine sample, add an equal amount of DNPH reagent in
test tube and mix it thoroughly.
Observation : A yellow precipitate indicates the presence of phenylpyruvate after
10 minutes.
Inference :PHENYLKETONURIA
B) Observation : A yellow precipitate indicates the presence of branched amino
acids or ketoacids.
Inference : MAPLE SYRUP URINE DISEASE
URINE BECOME DARK ON STANDING
Procedure: Take 5ml urine sample keep it for 10-12 hour.
Observation : If the urine is present with homogentistic acid urine
become black. On standing the urine is become alkaline by the
bacterial action with air. So in the alkaline urine the
homogentistic acid is oxidised in to black colour alkaptone bodies
Inference : ALKAPTONURIA
The metabolic defect is deficiency of homogentisate oxidase
BENEDICTS TEST:
Procedure: add 8 drops of urine sample in 5ml of benedict's reagent it. Boil it for
less than 2 minutes.
Observation : is strongly positive. A brick red precipitate is observed. Therefore
alkaptonuria comes under the differential diagnosis of reducing substance.
Inference: ALKAPTONURIA
Ammonical silver nitrate test
Procedure : take 3x3 square inch cut piece of filter paper. Mark 2 circle . One for
control and another is for test.
Patient sample in test and normal urine sample in control. Then add 5% freshly
prepared ammonical silver nitrate solution and observe.
Observation : The ammonical silver nitrate is converted to metallic silver it will
gives black colour. It is due to reduction of ammonical silver nitrate by
homogametic acid
Inference :ALKAPTONURIA
Cyanide nitroprusside test for homocysinuria
Procedure :to 5ml urine add 5 drops of conc. NH4OH and 2ml of NaCN
(5g/dl).keep for 10mnt.
Observation : sodium nitroprusside react with sulfhydryl compounds in
homocysteine and cysteine to produce red purple product.
Inference :HOMOCYSTENURIA
It is an inherited disorder of metabolism of the methionine due to deficiency of
cystathionine β synthase
Rothera’s test
Procedure : Urine sample saturate with solid ammonium sulphate.Add 1 ml of 5%
sodium niroprusside mix thoroughly. Now add con. Aqueous ammonia along the
side of the test tube.
Observation :A purple colour ring develops at the junction of two liquids.
Urine contain branched chain keto acid valine, leucine, isoleucine gives positive.
Inference: MAPLE SYRUP URINE DISEASE
• This is a metabolic disorder of branched chain amino acid.
• The urine of effected individuals smell like maple syrup or burnt sugar .
• It is due to the defect in enzyme branched chain alpha keto acid dehydrogenase
Mucic acid test for galactose
• Procedure: Take 6ml of urine sample and add 1ml of mucic acid reagent
Heat the test tubes in the water bath for 1- hour until solution is reduced to 2-3 ml.
Observation : Nitric acid yield saccharic acids(dicarboxylic acids).
• Formation of crystal at the bottom of the tube indicates a positive results.
Inference: GALACTOSEMIA
There could be deficiency of the three enzymes , galactosidase, 4-
epimerase and uridyl transferase
OBERMAYER TEST
Procedure : Add 5ml urine and 5ml obermayer indicator in a test
tube and mix it thoroughly. Add 2-3 ml of chloroform.
Observation :a blue colour formed at the bottom of tube indicates
the presence of indicant.
Inference :HARTNUP DISEASE
Absorption of aromatic amino acid from intestine as well as
reabsorption from renal tubules are defective ,so amino acid are
excreated through urine.
CALCULATION OF ENERGY CONTENT
OF DIFFERENT FOOD ITEMS
Calorific value of food
• Calorific value of food refers to the amount of energy
released by the food in the body on oxidation. Carbohydrate – 4 Kcal/g
Fats – 9 Kcal/g
• Expressed in calories/ kilocalories. Proteins – 4 Kcal/g
Alcohol – 7 Kcal/g
1 CALORIE – Heat
required to raise the
temperature of 1 g of
water through 1°C
Calculation of energy contents of food
Carbohydrate – 4 Kcal/g
Fats – 9 Kcal/g
Proteins – 4.2 Kcal/g
Alcohol – 7 Kcal/g
Calculate the energy contents from 200gm of carbohydrates, 40gm of fats and
30gm of proteins.
Carbohydreates = 200x4 =800
Fats =40x9=360
Proteins =30x4=120
total =800+360+126 = 1280kcal/g
Calculate the energy contents from 400gm of carbohydrates, 50gm of fats and
60gm of proteins.
Carbohydrates= 4x400 =1600
Fat = 9x50= 450
Proteins = 4x60=240
Total = 2290 kcal/g
Calculate the energy contents from 600 gm of carbohydrates, 60gm of fats and
70gm of proteins.
Carbohydrates = 4x600= 2400
Fats = 9x60=540
Proteins= 4x70=280
Total =3220kcal/g
