Phenylalanine-Tyrosine Disorders Branched-Chain Amino Acid Disorders Purine Metabolism

1. Amino Acid Disorders
Branched-Chain Amino Acid
Disorders
Purine Disorders
Date submitted: February 19, 2014
Section: MD3Y2-7
Name: Pagdanganan, John Kenneth
Clanor, Angelica
Bonto, Mae Willrose

2. Amino Acid Disorders
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Amino acid disorders w/ urinary screening
tests:
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Phenylketonuria
Tyrosyluria
Alkaptonuria
Melanonuria
Maple syrup urine disease
Organic acidemias
Indicanuria
Cystinuria
Cystinosis

3. Phenylalanine-Tyrosine Disorders
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Many of most frequently requested special urinalysis
assoc. w/ this metabolic pathway
Major inherited disorders:
PKU
● Tyrosyluria
● Alkaptonuria
Metabolic Defects: Melanoma
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4. Phenylketonuria
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Most well known aminoaciduria
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1/10k-20k births
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Can cause:
Severe mental retardation if undetected
● Fair complexions due to decreased prod of
tyrosine & melanin
Identified in Norway by Ivan Folling (1934)
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Peculiar mousy odour of urine of mentally retarded
child
High levels of keto acids, inlcuding
phenylpyruvate
Conversion of phenylalanine to tyrosine is disrupted

5. ●
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Absence of gene needed to prod.
Phenylalanine hydroxylase
Autosomal recessive trait: normal
heterozygous carrier
Newborn screening test: (+) PKU = eliminate
phenylalanine in diet (major constituent in milk)
= prevent excessive buildup in serum = avoid
mental retardation
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Alternate pathways develop as child matures
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Aspartame: high levels of phenylalanine

6. ●
Blood Test
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Initial screening
done b/f being discharged from hospital
Can be detected 4hrs after birth if cutoff is
lowered to 2mg/dL
May need to repeat test in early visit to
pediatrician
Slower rise in girls- more prone to escape
detection during early tests

7. ●
Microbial Inhibition Assay – Guthrie
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Most well-known blood test for PKU
P: phenylalanine counteracts action of beta-2thienylalanine, an inhibitor of B. subtilis
1. Blood from heelstick absorbed into filter
paper circles
2. Paper disks placed on CM streaked w/
B. Subtilis
(+) growth around paper disk
(-) no growth

8. ●
Urine Test
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Appears after 2-6 weeks in urine
Follow up procedure in questionable diagnostic
cases
Screening test to ensure proper dietary control
Monitoring dietary intake of pregnant women
lacking phenylalanine hydroxylase

9. ●
Ferric Chloride
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Urine tube test testing for phenylpyrvic acid
Nonspecific: reacts with other amino acids &
medications
False (+) produced by certain brands of
disposable diapers
Permanent blue-green colour
1. Place 1mL of urine in tube
2. Slowly add 5 drops of 10% ferric chloride
3. Observe colour

10. Tyrosyluria
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Tyrosinemia produces urinary overflow
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Inherited/Metabolic
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Since 2 reactions are directly involved in tyrosin
metabolism, urine may have:
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Excess tyrosine
Excess degraded products:
● P-hydroxyphenylpyruvic acid
● P-hydroxyphenyllactic acid

11. ●
Transitory Tyrosinemia
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Most frequently seen in premature infants
Caused by underdevelopment of liver fxn req.
to prod. enzymes needed for complete
metabolism
Rarely causes permanent damage
Difference in urinary test w/ PKU
● Green colour fades away quickly

12. ●
Acquired liver Disease
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Resembles transitory newborn tyrosyluria
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Leucine & Tyrosine crystals seen
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Hereditary Disorders
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Absence of enzymes needed in metabolic pathway
Serious & usually fatal
Liver & Renal Tubular Damage
Generalized aminoaciduria
3 types (all produce tyrosylemia & tysosyluria)

13. ●
Type 1:
Deficient in Fumarylacetoacetate hydrolase
(FAH)
● Generalized renal tubular disorder & progressive
liver failure
Type 2:
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Deficient in Tyrosine aminotransferase
● Corneal erosion; lesions of palms, fingers, &
soles of feet due to crystallization of tyrosine in
cells
Type 3:
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Deficient in P-hydroxyphenylpyruvic acid
dioxygenase
Mental retardation if dietary restrions of
phenylalanine & tyrosine not implemented

14. ●
Nitroso-naphtol Test
Urinary screening test for tyrosine & its
metabolites
● Nonspecific
● (+) orange-red = indicates need of further tests
1. Place 5 drops of urine in a tube
2. Add 1mL of 2.63N nitric acid
3. Add 1 drop of 21.5% sodium nitrite
4. Add 0.1mL l-nitroso-2-napthol
5.Mix
6. Wait 5 minutes
7. Observe colour
MS/MS screening test
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Available for Type 1 & 2

15. Melanuria
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2nd pathway of Tyrosine
Responsible for prod. of melanin, thyroxine,
epinephrine, protein & tyrosine sulfate
Major lab concern: Melanin
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Confers dark hair, eye, and skin colour
Deficieny: Albinism
Elevated: overproliferation of melanocytes =
Malignant Melanoma
Tumor cells secrete 5,6-dihydroxyindole =
oxidizes to melanogen, then melanin =
dark urine

16. ●
Melanin reacts w/:
1. Ferric Chloride: gray/black precipitate
2. Sodium Nitroprusside (nitroferricyanide): red
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Glacial acetic acid: prevents intereference from
creatinine & acetone
Melanin: green-black
Acetone: purple
Creatinine: amber
3. Ehrlich Reagent

17. Alkaptonuria
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1 of 6 inborn errors of metabolism
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3rd major defect in phenylalanine-tyrosine pathway
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Absence of gene needed to prod. Homogentisic acid
oxidase
Homogentisic acid accumulates in blood, tissue, urine
“alkali lover” - turns dark & alkali after standing in room
temp
Brown pigment deposited in body tissues (esp. ear);
deposit in catilage (arthritis)
Many develop liver & cardiac disorders

18. ●
Homogentisic acid reacts w/:
1. Ferric Chloride: transient deep blue colour
2. Clinitest: yellow precipitate
3. Alkali: darkening of urine (interference: large
amounts of ascorbic acid)
Silver nitrate & ammonium hydroxide: black
Read:
Spectrophotometer, Chromatography procedures

19. Branched-Chain Amino Acid
Disorders
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A methyl group branches from main aliphatic
carbon chain
1. accumulation of 1/more early amino acid
degradation products (maple syrup urine dse)
2. organic acidemias: accumulation of organic
acids further down metabolic pathway
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Ketonuria in newborn

20. Maple Syrup Disease
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Rare, Autosomal recessive trait, caused by IEM
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Involved: leucine, isoleucine, valine
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Absence of gene for oxidative carboxylation of keto
acids (alpha-ketoisovaleric, alpha-ketoisocaproic,
alpha-keto-beta-methylvaleric) = accumulation in
blood & urine
Failure to thrive after aprox. 1 week
Maple syrup odor (11th day): due to rapid
accumulation of keto acids
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REPORT! Prevent severe mental retardation &
death:

21. ●
Newborn screening: MS/MS
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2,4-dinitrophenylhydrazine (DNPH) reaction
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Most frequently performed urinary screening
(+) yellow turbidity or precipitate
Interferece: Large doses of ampicillin
Nonspecific
Treatment can be started based on odor, clinical
symptoms & positive DNPH test while
confirmatory prodecures are being done

22. ●
2,4-Dinitrophenylhydrazine Test
1. Place 1mL of urine in a tube
2. Add 10 drops of 0.2% 2,4-DNPH in 2N Hcl
3. Wait 10 minutes
4. Observe for yellow/white precipitate

23. Organic Acidemias
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Generalized symptoms:
Early severe illness
● Vomiting w/ Metabolic acidosis
● Hypoglycemia
● Ketonuria
● Increased serum ammonia
Most frequent disorders:
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Isovaleric acidemia
Propionic acidemia
Methylmalonic acidemia

24. ●
Isovaleric Acidemia:
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Sweaty feet odor of urine
● Caused by accumulation of
isovalerylglycine due to a deficiency of
isovaleryl coenzyme A in the leucine
pathway
Propionic & Methylmalonic acidemia:
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Errors converting isoleucine, valine, threonine,
methionine to succinyl coenzyme A
Immediate precursor to methylmalonic acid:
Propionic acid

25. ●
Newborn screening: MS/MS
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Isovaleric acidemia
Propionic acidemia
Methylmalonic acidemia
Urine test: Methylmalonic aciduria
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P-nitroaniline: (+) emerald green colour

26. Purine Disorders
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Lesch-Nyhan Disease
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Sex linked recessive
Massive excretion of uric acid crystals
Absence of Hypoxanthine guanine
phosphoribosyltransferase
Severe motor defects, mental retardation,
tendency towards self-destruction, gout, renal
calculi
First 6-8 months: normal development
First symptom: (uric acid) orange sand in
diapers

31. Source:
Urinalysis and Body Fluids, 5th edition
by Susan King Strasinger & Marjorie Schaub Di
Lorenzo