This is a powerpoint file of an MBBS practical class taken by Dr. Karthikeyan Pethusamy at All India Institute of Medical Sciences - NewDelhi. Disclaimer: The views expressed here are of the author only not of the institution.

1. SCREENINGTESTS FOR INBORN
ERRORS OF METABOLISM
FIRST MBBS PRACTICAL

2. Objectives
• To understand the importance of screening test
• To define the inborn errors of metabolism and their
biochemical aspects
• To know the principle of the various screening tests done

3. What is “SCREENING”?
Disease
onset
First
possible
detection
Final
critical
point
Usual
time of
diagnosis
A (Usually poor)
B (Better)
Outcome
Lead time
Screening is a process of identifying apparently healthy people who may
be at increased risk of a disease or condition.

4. Screening tests and Diagnostic tests
Screening test
• Apparently healthy
• More sensitive, less specific
• Results are suggestive and
only gives hints
• Positive result not a basis
for treatment
• Economical
Diagnostic test
 Sick or with overt indications
 More specific
 Results are definitive
 Positive test may be a basis
for treatment
 Comparatively expensive

5. Metabolism
• Metabolic processes – All chemical reactions that occur in
the living cell
– Anabolism
– Catabolism
• Metabolic pathways
• Series of enzyme controlled reactions leading to the
formation of a product
• Each new substrate is the product of the previous reaction

6. A
B
C
D
Enzyme a
Enzyme b
Enzyme c
EF
Metabolic Pathway

7. Inborn Errors of metabolism
Inborn Errors of Metabolism (IEM) comprise a
group of disorders in which a single gene defect
causes a clinically significant block in a metabolic
pathway resulting either in accumulation of
substrate behind the block or deficiency of the
product.

8. A
B
C
D
Enzyme a
Enzyme b
Enzyme c
EF
Decreased synthesis of
important metabolite
Accumulation of Substrate
Conversion of substrate
into other, often
undesirable compounds
Inborn Errors of metabolism

9. Inborn Errors of metabolism
• Rare in occurrence
• Detected most frequently at infancy
• Some are fatal at early age

10. Classes of Inborn Errors of metabolism (IEM)
• Disorder of carbohydrate metabolism
– Galactosemia
– G6PD deficiency
• Aminoacidopathies
– Phenylketonuria
• Urea cycle defects
– Hyperammonemia
– Citrullinemia
• Organic acidemias
– Alkaptonuria

11. Glucose-6-phosphate dehydrogenase (G6PD)
deficiency
Glucose-6-phosphate 6-Phosphogluconolactone
Glucose -6- phosphate dehydrogenase
NADP+ NADPH + H+

12. Glucose-6-phosphate dehydrogenase (G6PD)
deficiency
• G6PD is the enzyme catalysing the first oxidative,
irreversible, NADPH producing step of HMP shunt
• NADPH is important for maintaining the glutathione in
reduced state.

13. Role of glutathione in maintaining
membrane integrity

14. Glucose-6-phosphate dehydrogenase (G6PD)
deficiency
• Increased oxidative stress eg: drugs in G6PD deficiency
patients unable to generate enough NADPH - > oxidative
damage - > decreased life span of RBC -> hemolytic anemia
• Pattern of inheritance – X linked recessive

15. G6PD deficiency (Screening test)
a) Bernstein Method:
Fresh blood is haemolysed and to it freshly prepared indophenol
dye + NADP + G-6-P + Phenazine Methosulfate is added.
Glucose-6-phosphate 6-Phosphogluconolactone
Glucose -6- phosphate dehydrogenase
NADP+
NADPH + H+
indophenol dye NADPH
+ H+
+
Reduced form
(Colourless)
+ NADP+
Phenazine Methosulfate
Normal – 30min -1hr
G6PD deficiency - > 2hrs

16. G6PD deficiency (Screening test)
b) Methylene blue Reduction Method:
Hemolysed Blood
Hb2+
Hb3+
NaNO2
Methylene Blue
NADP+
NADPH+H+
Dark Red Chocolate Brown
G6PD deficiency – Persistence of chocolate brown color

17. Galactosemia
• Due to deficiency of enzymes:
 Galactose -1 phosphate uridyl transferase
 Galactokinase
UDP – galactose – 4- epimerase
• Increased galactose levels in blood and urine

18. Galactose metabolism

19. • Galactose is converted to galactitol by aldose reductase -
Cataract
• Pattern of inheritance – Autosomal recessive

20. Galactosemia
(Screening tests)
• Demonstration of reducing sugar in urine
• Beutler enzyme spot test:
Galactose -1- phosphate uridyl transferase activity is
monitored on blood dried on filter disc with the aid of
Galactose1Po4,UDP-glucose, NADP+
phosphoglucomutase,
glucose -6- phosphate dehydrogenase followed by
measurement of the absorbance of reduced NADP+
under UV
light.

21. Galactosemia
(Beutler enzyme spot test)
Galactose-1-phosphate
Glucose-1-phosphate
Glucose-6-phosphate
6-Phosphogluconolactone
Galactose -1 phosphate uridyl transferase
Phosphoglucomutase
Glucose -6- phosphate dehydrogenase
NADP+
NADPH + H+

22. Beutler enzyme spot test

23. Mucopolysaccharidoses
 Deficiency of a group of enzymes which degrade three
classes of mucopolysaccharides: dermatan, Keratan and
Heparan sulphate
 Excretion of excess mucopolysaccharides in the urine

24. Mucopolysaccharidoses

25. Muco-polysaccharidoses
• Screening test used is Alcian blue spot test
• Alcian blue is a copper phthalcyanin dye and contains
positively charged groups capable of salt linkage with certain
polyanions (sulphate and carboxyl radicals of the acid
mucins)
• 10ml urine is dried on filter paper and Alcian blue is added
• Urine appears as a blue spot on a white back ground

26. Alcian blue test

27. Inborn Errors of metabolism (IEM): definitive
diagnostic tests
 Measurement of the enzyme levels/activity in the cells
(leukocytes, fibroblasts etc.)
 Prenatal diagnosis – measurement of the enzyme
levels/activity in the cultured cells from the amniotic fluid
 Molecular genetic techniques (DNA or RNA based) using
chorionic villus sampling or amniocentesis

28. Phenylketonuria
• Most common inborn error of metabolism in Caucasian
population (1 in 10,000 births)
• Accumulation of phenylalanine and its metabolites
• Arises from defects in phenylalanine hydroxylase itself ( classic
phenylketonuria or PKU) or
– dihydrobiopterin reductase
– dihydrobiopterin biosynthesis
• Pattern of inheritance – autosomal recessive

29. Phenylalanine metabolism
NADP+
NADPH+ H+
Tetrahydrobiopterin Dihydrobiopterin
L-Phenylalanine L- Tyrosine
O2 H2O
Dihydrobiopterin
reductase
Phenylalanine
hydroxylase

30. Phenylketonuria
NADP+ NADPH+ H+
Tetrahydrobiopterin Dihydrobiopterin
L- Phenylalanine L- Tyrosine
Phenylpyruvate
Phenylacetate Phenyl-lactate
Phenylacetylglutamine
Substrate accumulation
Decreased synthesis
of important
metabolite
Conversion of
substrate into other,
often undesirable
compounds
O2 H2O
Dihydrobiopterin
reductase
Phenylalanine
hydroxylase

31. Irreversible damage to the nervous
system
• Accumulation of neurotoxic metabolites
• Deficiency of certain product will decrease
the synthesis of neurotransmitters
• Defect in the protein synthesis

32. Phenylketonuria
• Learning disabilities, abnormal EEG, seizures
• Intellectual disability, irregular motor
functioning
• Mousy/musty odor of urine and sweat
• Increased levels of phenylalanine in serum

33. Phenylketonuria Screening Tests
a. Guthrie’s bacterial inhibition test:
• Bacillus subtilis is used in this screening test .
• Bacillus subtilis requires phenylalanine for growth in
culture
• In presence of phenylalanine antagonist ( β-2
thienylalanine) in culture media– bacteria stops
growing

34. • Blood from normal infant is added – no growth of
bacteria
• Blood from phenylketonuria patient is added – bacteria
proliferates since phenylalanine is not metabolized and
present in high amounts.

36. Phenylketonuria Screening Tests
b. Ferric chloride test:
Take 5 ml of fresh urine sample, add 3-4 drops of ferric chloride
solution – phenylpyruvate gives green/blue colour

37. Phenylketonuria Screening Tests
Dinitrophenylhydrazine (DNPH) test:
Take 2 ml of urine and add an equal amount of DNPH reagent
and mix - wait for 10 mins: appearance of yellow precipitate
implies the presence of keto acids: phenylpyruvate.
DNPH + Phenylpyruvate = Hydrazone (Yellow ppt)

38. Alkaptonuria
• First disease to be identified as inborn error of
metabolism
• Striking feature - darkening of urine on standing
• Deficiency of homogentisate oxidase
• Pigmentation of connective tissue (Ochronosis) and
arthritis.
• Pattern of inheritance – autosomal recessive

39. Tyrosine metabolism
L-TyrosineL-Tyrosine HomogentisateHomogentisate
MaleylacetoacetateMaleylacetoacetateFumarylacetoacetateFumarylacetoacetateAcetoacetateAcetoacetate
Acetate + Acetyl -CoAAcetate + Acetyl -CoA
Homogentisate oxidaseHomogentisate oxidase
p – hydroxyphenylpyruvatep – hydroxyphenylpyruvate

40. Alkaptonuria
L-Tyrosine p – hydroxyphenylpyruvate Homogentisate
MaleylacetoacetateFumarylacetoacetateAcetoacetate
Acetate + Acetyl -CoA
Homogentisate oxidase
Brownish
black
pigment
O2
Polyphenol
oxidase
Benzoquinone
acetate
Polymerizes
and binds to
connective
tissue
Ochronosis

41. Normal urine Urine from alkaptonuria patient
Symptoms of alkaptonuria
Patients may display painless bluish darkening of the outer ears, nose and whites of the
eyes.

42. Alkaptonuria
(Screening tests)
a. Benedict’s reagent – brown colour
b. Ferric chloride test – immediate black colour
c. Saturated silver nitrate test – immediate black colour
d. X-ray lumbar spine- bamboo like appearance

43. X-Ray Lumbar spine
Alkaptonuria Normal

44. Maple syrup urine disease
 Deficiency of the enzyme – branched chain α-keto acid
dehydrogenase complex.
 Block in the metabolism of branched chain amino acids
(valine, leucine, isoleucine)
 Plasma and urinary levels of valine, leucine, isoleucine, α-
keto acids and α-hydroxy acids are raised
 Smell of maple syrup (burnt sugar) in urine due to keto-
acids
 Pattern of inheritance – autosomal recessive

45. Maple syrup urine disease
L-leucine
α-Ketoisocaproate
Isovaleryl-CoA
β-Methylcrotonyl-CoA
L-valine
α-Ketoisovalerate
Isobutaryl-CoA
Methacrylyl-CoA
L-Isoleucine
α-Keto - β -methylvalerate
Tiglyl-CoA
α -Methylbutaryl-CoA
branched chain α-keto acid dehdrogenase complex

46. Maple syrup urine disease
L-leucine
α-Ketoisocaproate
Isovaleryl-CoA
β-Methylcrotonyl-CoA
L-valine
α-Ketoisovalerate
Isobutaryl-CoA
Methacrylyl-CoA
L-Isoleucine
α-Keto - β -methylvalerate
Tiglyl-CoA
α -Methylbutaryl-CoA
branched chain α-keto acid dehydrogenase complex

47. Maple syrup urine disease
(Screening tests)
• Guthrie’s bacterial inhibition test: 4-Azaleucine
antagonist of L-leucine
• Ferric chloride test – greenish grey colour
• DNPH test is positive