METABOLIC SCREENING TESTS FOR INBORN ERRORS OF METABOLISM

LABORATORY PROTOCOL

SAMPLE COLLECTION AND PROCESSING

RANDOM URINE SAMPLE

(50ml for complete & 10ml for MPS)

PRESERVATIVE -1ml 6N HCl for 10 ml of urine

Centrifuge at 5000 Rpm for 15 min

Collect the supernatant

Proceed with chemical tests & Chromatography

Observe for physical appearance

Colour

Odour

Clarity

PH

Bloody contaminants

URINE MICROSCOPY

Reagents:2% Aqueous Methylene Blue, 1% Toluidine Blue in 1N acetic acid Method: The test should be performed on fresh urine sample.(renal cells will be destroyed otherwise).Centrifuge the urine and decant the supernatant fluid. Add 2 drops of Stain to the sediment. Examine under high power microscopy after 5 to 10 minutes.

Cellular Pathology: With Methylene Blue stain, Metachromatic granules show

blue within renal epithelial cells. In older urine the granules are seen extracellularly as the cells will be ruptured. A positive test requires more than 10% cells to be stained and indicates Sulfatide lipodystrophy (Metachromatic Leukodystrophy). A similar test with 1% Toluidine Blue in1N Acetic acid gives positive test for Mucopolysaccharidosis. Granules in this stain purplish and are smaller. Presence of other cells should be noticed (Hereditary nephritis in Alport’s disease with deafness- IEM of Proline metabolism) Note: Uric acid crystals dissolve in alkaline pH and Phosphates in acidic pH. Uric acid crystals are also seen in normal urine.

1. BENEDICT’S TEST

PROCEDURE OBSERVATION INFERENCE 2ml Benedict’s reagent + 0.2ml urine .Place test tubes in boiling water bath for 3 minutes .Take it out and allow to cool to room temp.

Appearance of precipitates of various colours indicates positive test- green, yellow, red, brick red

Reducing sugars are present

Reagent preparation : A: 17.3 gm Copper sulphate (CuSO4 5 H2O) in 200ml water

B: Dissolve 173gm Sodium Citrate in 500 ml water (heat if necessary).Add 100 gm

anhydrous Sodium Carbonate and dissolve with stirring. After cooling mix “A” & “B”

Note : White precipitate -Phosphates may be present

2. NINHYDRIN TEST

PROCEDURE OBSERVATION INFERENCE To 1 ml of urine ,add 10 drops of ninhydrin .Heat to boiling

Appearance of Blue colour

Free amino groups are present .

Principle: Ninhydrin reacts with free Alpha amino groups of proteins to give blue to purple coloured complex called Ruhemann’s Purple Reagent preparation :

1. 0.2% Ninhydrin Solution

PROCEDURE OBSERVATION INFERENCE

Add three drops of urine to 1 ml of reagent in a clean test tube. Mix .Observe the colour after 3 – 4 minutes

Appearance of Blue or purple colour

Excessive Aminoaciduria is present

False positive: Concentrated urine, High Ammonia in urine Principle: Ninhydrin reacts with free Alpha amino groups of proteins to give blue to purple coloured complex called Ruhemann’s Purple Presence of single amino acid in excess without overall increase in amino acids may not be detected. Reagent preparation :

1. 1gm of Ninhydrin in 500 ml of 95% Ethanol. Reagent should be kept in Dark brown bottle in refrigerator

3.DINITRO PHENYLHYDRAZINE (DNPH) TEST

PROCEDURE OBSERVATION INFERENCE Centrifuge the urine(5000rpm /15 min) .Add 0.2ml clear urine to test tube.0.8 ml of DNPH reagent. Mix .Allow to stand for five minutes. If turbidity is equivocal , add another 0.4 ml of DNPH reagent

Appearance of turbidity or yellow white precipitate

α Keto acids are present

Reagent preparation: 200 mg DNPH in 100 ml 2N HCl. Store at ambient temperature stable for 3 months

4.FERRIC CHLORIDE TEST

PROCEDURE OBSERVATION INFERENCE

Take 1ml of urine (T) and 1ml of reference standard (C) in two test tubes respectively. Add 0.2 ml of ferric chloride reagent to both the test tubes and mix. Note the colour change if occurs immediately & after 2-3 minutes

Appearance of Change in colour ( blue, green, greenish grey , purple, deep green

Aromatic amino acids present

Reagent preparation: Dissolve 10 gm ferric chloride (anhydrous) in water and make up to 100 ml. Store in dark bottle at 40C.

5.METHYL MALONIC ACID TEST

PROCEDURE OBSERVATION INFERENCE Label 2 test tubes as T (test)& C (Control) .Add 0.1ml of urine to test tube & 0.1 ml of Methyl malonic acid standard to tube “C”respectively. Add 1ml of p- Nitroaniline reagent to each of the tubes and mix .Add 0.25 ml of Sodium nitrite solution(0.5 %) to each of the tubes & mix .Allow tubes to stand at room temperature for a minute.Add 1.5 ml of Sodium acetate buffer,PH 4.3 & mix.Place the tubes in boiling water bath for 3 minutes ,add 0.3 ml of N/8 Sodium Hydroxide & mix

Appearance of emerald green colour

Methyl malonic acid present(60 mg/dl)

Reagent preparation :

1. MMA reference std(0.025 M): 147.5 mg Methyl malonic acid(MW 118.1) in 50 ml of water and add a 6N HCl to render it acidic. Store in refrigerator at 40C.Stable for six months

2. P-Nitroaniline reagent : Dissolve 100mg p-Nitroaniline in 100 ml of 0.16 N 3. HCl .Store in dark bottle at room temperature. 4. Sodium Nitrite (0.5%) :Dissolve 0.5g of Sodium nitrite in water & male up to

100 ml .Stable for 2 months in refrigerator. 5. Sodium acetate buffer 1 M pH 4.3 :Dissolve 13.6 gm Sodium acetate

trihydrate in distilled water &adjust the pH to 4.3+0.02 make up the final volume to 100 ml .Keep it in refrigerator at 40C.Stable for 2-3 months

6. NaOH (8N) : Dissolve 32 gm of NaOH in water, while keeping it cold in a ice bucket, and adjust volume to 100 ml. Store in plastic bottle with screw cap at room temp.

False positive: Children on Valproic acid treatment for seizures

6.NITROSONAPHTHOL TEST

PROCEDURE OBSERVATION INFERENCE To a test tube add 1 ml nitric acid ,0.1 ml of sodium nitrite solution and 0.1 ml of Nitrosonaphthol solution and mix Add 0;.3 ml of urine and mix. Wait for five minutes .

Appearance of orange to deep red colour within five minutes

Tyrosine and its metabolite present

Reagent preparation:

1. Nitric acid(2.6N): Add one volume Conc HNO3 to 5 volumes of water 2. Sodium nitrite solution : Dissolve 2.5 g Sodium nitrite in water &

make up to 10 ml .Store at 40C. 3. 2- Nitrosonaphthol reagent: Dissolve 10 mg of Nitrosonaphthol in

100 ml ethanol(95% v/v) .Store at 40C.

False positive : Patients with GI disorders due to hydroxyl phenyl acetic acid due to bacterial overgrowth in intestine.

7.SULPHURIC ACID TEST FOR INDOLE DERIVATIVES

PROCEDURE OBSERVATION INFERENCE Add 0.5ml of reagent to 5 ml of urine. Observe the colour after ten minutes

Appearance of strong red or violet colour indicates positive test

Hydrindic acid is present

False positive : Drugs like Phenothiazines Reagent preparation: Conc Sulphuric acid

8.ISATIN TEST

PROCEDURE OBSERVATION INFERENCE Dip some filter paper (Whatmann’s No 3 or Equivalent)into reagent A.Allow it to dry at room temperature.Add one drop of urine and allow it to dry at 100o

C for ten minutes.Dip in reagent B and wash it with water.

Appearance of deep blue colour of urine spot indicates a positive reaction

Proline is present in excess

Reagent preparation: A: 20 mg Isatin ,96 ml Acetone,4 ml of Glacial acetic acid.The reagent will be stable for three months if kept in refrigerator B:1N HCl

9.ROTHERA’S TEST FOR KETONE BODIES

PROCEDURE OBSERVATION INFERENCE Take 1 cm length solid Ammonium Sulphate in a test tube .Add 5 ml urine .Shake to dissolve and saturate. Some solid should remain at the bottom .Add 1 drop of freshly prepared solution of Nitroprusside solution. Mix well. Add 0.5 -1 ml of liquor Ammonia gently along the sides of the test tube.

Appearance of purple ring indicates positive reaction

Ketone bodies are present

Reagent preparation:

1. Sodium Nitroprusside solution : Freshly prepared 5% solution 2. Solid Ammonium Sulphate 3. Liquor Ammoni

10. EHRLICH’S ALDEHYDE TEST FOR PORPHOBILINOGEN

PROCEDURE OBSERVATION INFERENCE

Add 1ml of reagent to 5 ml of fresh urine Examine for colour after ten minutes If positive add 5ml of Chloroform with shaking

Appearance of pink colour indicates positive reaction Pink colour is extracted into chloroform layer

Porphobilinogen/Urobilinogen present

Urobilinogen present

(porphyrins are not extracted into chloroform layer)

False positive: Indoles in urine can give positive test Reagent preparation:

1. Ehrlich’s Reagent: 2%p-dimethylaminobenzaldehyde in 2N HCl

11.O-TOLUIDINE TEST FOR COPPER

PROCEDURE OBSERVATION INFERENCE Put 2 drops of urine on thick filter paper(Whatmann’s no 3 or equivalent).Allow it to dry and then add one drop of freshly made reagent.

Appearance of blue colour within 30 seconds suggests positive reaction

copper is present in excess

Wilson’s disease to be considered

Reagent preparation: O-Toluidine 0.1 gm, Ammonium Thiocyanate 0.5 gm, Acetone 5 ml. Mix just before use

12.Tests for Mucopolysaccharidosis

1. Spot test for MPS

PROCEDURE OBSERVATION INFERENCE Mark three spots on Whatmann filter paper no 3 with lead pencil .Spot 5μl urine on one spot .10μl on another and 5μlf of reference std on another spot & dry the spots.Stain the spots by placing in Toluidine blue solution for 45 seconds. Rinse the paper with water to remove the excess stain. Destain for 10 min in washing solution

Appearance of Purplish or bluish spot remaining after destaining is positive test Absence of spot or Metachromatic ring is negative Positive with 10μl but negative with 5 μl is suspicious

MPS is present

Reagent preparation:

1. Toluidine blue O Stain : 200mg Toluidine Blue O (MW 305.8) in 100 ml of 80% Acetone

2. Washing Solution : Water :Methanol:Glacial acetic acid(200:20:1 v/v) 3. Chondroitin Sulfate ( ref Std) : Dissolve 10mg of ChondroitinSulphate

B in 10 ml water , Store in refrigerator.

Tests for Mucopolysaccharidosis

4. Cetyl Pyridinium Chloride

Citrate Turbidity Test

PROCEDURE OBSERVATION INFERENCE Centrifuge the urine at 5000 rpm for 10 min. Add 1 ml of urine to each of two test tubes labeled “T” & “C”. Add 1ml of ref std solution of Chondroitin sulphate to test tube labeled as “RS” .Add 1ml of CPC reagent to tube “T” and “RS” and mix. Allow the tubes to stand at room temperature for at least 30 min.

Appearance of turbidity in “T” as in “RS”

MPS is present

Reagents:

1. Citrate buffer(PH 4.8): Add 15.88gm of Tri sodium Citrate in water and adjust the PH to 4.8and make the volume to 1 litre

2. Cetyl Pyridiunium Chloride reagent (0.1 gm%) : Dissolve 100 mg CPC (mol wt 340)in 100 ml of citrate buffer .Both reagents are stable for at least 6 months at room temp.

Chromatography For Amino Acids

Reagents:

1. Reference standards of amino acids:

Stock std: Prepare equimolar solution (10mM) of each individual amino acids in Methanol: Water (8:2V/V) mixture.

Standard for use: Dilute 1:100 V/V of stock standard to a final concentration of 0.1mM with Methanol: Water mixture .5μof this reference standard would be equivalent to 0.5nmoles of individual amino acids which is easily detectable by ninhydrin method on TLC plate. i.e, 100μl of 10mM stock std + 900μl of methanol: water (80:20) mixture.

Std mixture: Take 200μl of six amino acids (10mM stds) and mix .Add 800μl of Methanol: Water (8:2) mixture. Cysteine Histidine Alanine Mixture 1 Tyrosine Methionine Leucine Cystine Lysine Hydroxyproline Mixture 2 Glutamic acid Valine Phenyl alanine

Aspartic acid Arginine Glycine Mixture 3 Tryptophan Proline Isoleucine

2. Silica Gel: 50 gms of silica in 100 ml of distilled water. 3. Solvent system: n-Butanol: Acetic acid: Water =

12:3:5(240:60:100) 4. Ninhydrin stock solution :1.4 gms Ninhydrin+ 10ml Acetone+

n-Butanol-10ml 5. Ninhydrin working solution: 2.5 ml of stock solution + 25 ml

of solvent. Sample Preparation: Take 1 ml of urine in eppendorf tube .Add 1ml of Methanol. Centrifuge (5000rpm for 15 min).collect the supernatant.

Procedure for Paper chromatography:

Saturate the chromatography chamber and whatman paper by keeping the solvent in the chamber overnight

Spot 5µ each of urine/plasma and reference standards on

to corresponding labeled lines on whatman filter paper and dry the spots with a hair dryer.

Place the paper in solvent tank and allow the solvent to ascend (4-6 hrs) till 2 cm from the top.

Take out the paper and dry it. Spray with ninhydrin reagent, dry it using dryer or keep it in hot air oven.

Look for spots in sample lanes corresponding to the Rf values of reference std. Note: wear gloves before touching the filter paper.

Procedure for Thin Layer chromatography:

Spot 5μl of Urine extract (1μl of urine) and reference standards on the correspondingly labeled lines. Distance between thw spots should be 2 cm atleast.

Activate TLC plate at 600 C for 10 min

Place the TLC Plate in Solvent tank and allow the solvent to ascend till 2 cm from the top of thin layer. Take out the TLC plate and allow drying at room temp(10-15 min)

Spray with ninhydrin reagent; Allow to dry at room temp.

Transfer the plate to hot air oven set at 1000C

Look for the spots in the sample lanes corresponding to the Rf values of reference standard.

Rf= Solute front/ Solvent front

Sl No Amino Acid M W 10mMStd in 5 ml

Solute front

Rf(Solvent front 13.7cm)

1 L-Aspartic acid 133.1 6.65mg 3.4 0.248 2 L-Asparagine 132.1 6.6mg 2.1 0.153 3 Arginine 210.7 10.53mg 2.7 0.197 4 Glycine 75.17 3.75mg 3.3 0.240 5 Glutamine 146.1 7.30mg 2.8 0.204 6 L-Glutamic acid 147.1 7.36mg 4.9 0.357 7 L-Cystine 240.3 12.0 mg 1.0 0.072 8 L-Cysteine 121.2 6.06mg 0.8 0.058 9 Isoleucine 131.2 6.56mg 8.6 0.627 10 Hydroxyproline 131.1 6.56mg 3.4 0.248 11 L-Histidine 10.48mg 2.3 0.167 12 L-Alanine 89.09 4.45mg 4.7 0.343 13 L-Tyrosine 181.2 9.06mg 6.7 0.492 14 L-Lysine 182.6 9.13mg 2.4 0.176 15 Methionine 141.2 7.46mg 7.3 0.536 16 L-Leucine 131.2 6.56mg 9.3 0.683 17 L-Tryptophan 204.2 10.21mg 7.1 0.522 18 L-Threonine 119.1 5.96mg 4.3 0.316 19 L –Serine 105.1 5.26mg 3.1 0.228 20 L Proline 115.1 5.76mg 4.7 0.345 21 L-Valine 117.1 5.86mg 7.2 0.529 22 Phenylalanine 165.2 8.26mg 7.5 0.551 23 Ornithine

Leu(0.683)

Ile(0.627)

Phe(0.551)

Met(0.536)

Val(0.529)

Trp(0.522)

Tyr(0.492)

Glu(0.357)

Pro(0.345)

Ala(0.343)

Thr(0.316)

Hyp. Asp(0.248)

Gly(0.240)

Ser(0.228)

Gln(0.204)

Arg(0.197)

Orn(0.18)

Lys(0.176)

His(0.167)

Asn(0.153)

Cystine(0.072)

Cysteine(0.058)

Chromatography For Carbohydrates

Reagents:

1. Reference standards of Sugars:

Standard: 100mg (10g/L) each of glucose, Galactose, Xylose, Lactose and Fructose in 10 ml water (isopropanol).1.5μl contains 5μg of these sugars.

Std Mixture: 1ml each of individual sugars solutions mixed. 2. Silica Gel: 50 gms of silica in 100 ml of distilled water. 3. Solvent system: n-Butanol: Acetic acid: Water =

75:25:6(225:75:18).Water content if less than 5 tails and more than 6 beards. (ref: varley 5th ed)

4. Spraying agent : 6.2 gms of Phenylamine + 19.2 ml of Acetone + 2 ml of Aniline + 2ml of Orthophosphoric acid Filter and use the filtrate for spraying.(ref:workshop protocol) Or First prepare Diphenylamine reagent . 250mg of Diphenylamine + 25 ml acetone +250µl aniline. Above reagent 9 ml + O-Phosphoric acid 1ml. Or Mix 10 volumes of solution of 10 ml/L of aniline and 10g/L of diphenylamine in acetone with 1 volume of orthphosphoric acid.(sp.gr 1.75)(ref: varley 5th ed)

Or 1g Diphenylamine+1ml Aniline+100 ml Acetone.Mix. Add 85% O-Phosphoric acid 10ml drop wise with gentle stirring. Sample Preparation: Take 1 ml of urine in eppendorf tube .Add 1ml of Methanol. Vortex mix .Centrifuge (5000rpm for 15 min).collect the supernatant.

Procedure for Thin Layer chromatography:

Spot 5μl of Urine extract (1μl of urine) and reference standards on the correspondingly labeled lines.

Activate TLC plate at 600 C for 10 min

Place the TLC Plate in Solvent tank and allow the solvent to ascend till 2 cm from the top of thin layer. Take out the TLC plate and allow drying at room temp(10-15 min)

Spray with spraying agent; Allow to dry at room temp.

Transfer the plate to hot air oven set at 1000C

Look for the spots in the sample lanes corresponding to the Rf values of reference standard.

Xylose-0.64(blue)

Glucose-0.53(blue)

Fructose-0.51(Pink brown)

Galactose-0.5(blue)

Lactose-0.25(blue)

Protocol for Alkaptonuria

(Additional tests)

1. 10% NaOH /5 % Ammoniacal AgNO3: Urine turns black

2. Chromatography for homogentisic acid Solvent: Butanol:Acetic acid: water- 4:1:5

3. Spraying agent: Folin phenol reagent followed by 10% Na2CO3

Or

10% Ammoniacal silver nitrate

Bial’s Test for Pentosuria Bial’s reagent : 300mg Orcinol is dissolved

in 100ml of Conc Hcl .Add 5 drops of 10% FeCl3 solution. Procedure:Mix 5 ml of Bial’s reagent with 0.5

ml of Sugar solution. Heat to boil. Observation: Pentoses:Green colour. Fructose:Red colour

Mucic Acid Test for Galactose

Procedure: 1 ml of sugar solution + 1ml of conc HNO3

.Boil for 1hr or till the acid vaporizes in boiling water bath .Cool and look for crystals

Inference: Appearance of precipitate

or Broken glass like crystals indicates positive test