DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404

Phenylketonuria
 Autosomal recessive
 Abnormality of the phenylalanine hydroxylase system
 Phenylalanine hydroxylase: the enzyme most commonly affected
 Other 3 % of the cases: enzymes responsible for co-factor
tetrahydrobiopterin are abnormal
 Several different inherited deficiencies having similar biochemical
and clinical consequences
 Result of disorder:
 Phenylalanine cannot be converted to tyrosine ⇒⇒ phenylalanine
accumulates in the plasma,
 And is excreted in the urine with its metabolites:
♦ Phenylpyruvic acid (a phenylketone)

 Mousy odour of urine

 Autosomal recessive

Clinical features of phenylketonuria

 Normal blood phenylalanine levels are (mean +/- SD) :
 58 +/- 15 µmoles/L in adults
 60 +/- 13 µmoles/L in teenagers
 62 +/- 18 µmoles/L in childhood
 In the newborn, the upper limit of normal is 120 µmoles/L (2 mg/dl)
(Scriver et al., 1985; Gregory et al., 1986)
 Untreated classical PKU: blood levels as high as 2.4 mM/L
 Irritability, feeding problems, vomiting, and fits during the first few
weeks of life
 Mental retardation developing at between 4 – 6 months, with
psychomotor irritability
 Often generalized eczema
 A tendency to reduced melanin formation because of reduced production
of tyrosine
 Many patients are paled skinned, fair haired and
 Blue-eyed

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Diagnosis:
Measure phenylalanine concentration in the blood taken from heel prick

Microbiological Guthrie test is only suitable for mass screening and should

be performed 4 days after birth when infant is taking milk

If too early, infant’s enzyme system not fully developed and test may show

false negative.
Repeat if test show positive result

Guthrie Test:
 For the screening of newborn infants for phenylketonuria (PKU)
 A microbiological assay for the presence of phenylalanine,
phenylpyruvate, and phenyllactate in blood or urine
 Phenylalanine, phenylpyruvate, phenyllactate: compounds present in
excess in the blood or urine of patients with PKU
 The test uses the growth of a strain of bacteria on a specially-prepared
agar plate as a sign for the presence of high levels of phenylalanine,
phenylpyruvate, and/or phenyllactate
 The compound B-2-thienylalanine will inhibit the growth of bacterium
Bacillus subtilis (ATCC 6051) on minimal culture media
 To prepare the sample for application, a small amount of blood (from a
heel puncture) or urine (from a diaper) is applied to a piece of filter paper
 A small disc is punched from the center of the spot of blood or urine, and
the disc is applied to the surface of a seeded, minimal-medium agar plate
that contains added beta-2-thienylalanine.
 If the sample contains phenylalanine, phenylpyruvate, and/or
phenyllactate then these compounds will diffuse into the agar medium
 If concentration high enough (as with the excess levels seen with PKU),
bacteria will grow under the disc, but not elsewhere
 Generally overnight incubation is enough to determine whether
phenylalanine, phenylpyruvate, and/or phenyllactate are present in
unusual concentrations in blood or urine

Managing PKU
 Aim of management: to lower plasma phenylalanine concentration by diet
restriction
 A reduced-protein diet should be consistently followed throughout life
 All natural sources of protein contain too much phenylalanine for children
with PKU
 PKU diets: free of meat, eggs, nuts, or bread and wheat products

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  Regular blood tests to measure phenylalanine levels:
 From ages 1 – 12, testing should be done twice a month;
 After age 12 and throughout adulthood, testing should be done once a
month
 High levels of phenylalanine in teens and adults negatively affect IQ
(intelligent quotient) and cognitive functions such as awareness,
knowledge, thinking, learning, and judgment
 It is difficult, expensive, and tedious for
 Both parents and child
 Individuals should be monitored especially if they plan to be pregnant
 Phenylalanine is an essential amino acid
 Deficiency in phenylalanine ﾁ¨ repercussions: impaired growth rate,
eczema, mental retardation
 Tyrosine: the immediate metabolite of phenylalanine ⇒ tyrosine
supplementation necessary
 Tyrosine which is not an essential amino acid becomes the essential
metabolite in this case
 Dietary supplementation can be life long

Tyrosinemia
 Rare, devastating disease
 Autosomal recessive
 Children with tyrosinemia are unable to completely metabolise the amino
acid tyrosine
 Buildup of tyrosine and its metabolites, causing a variety of symptoms

Tyrosinemia Type 1
 Presents as severe liver disease at less than six months of age
 Untreated: affected infant dies of liver failure within weeks / months of
onset of symptoms
 Chronic form: progressive, cirrhotic liver disease, Fanconi-like renal
syndrome, rickets, growth failure, repetitive bouts of neorologic crises ----
death before the age of 10
 Incidence is 1:100,000 to 1:120,000 live births in U.S. and Europe
 Most common in French-Canadians or Scandinavians
 Highest incidence is in Quebec = 1:1,846 live births
 Absence of enzyme fumarylacetoacetate hydrolase
 Enzyme deficiency ﾁ¨ fumarylacetoacetate ↑↑
 Fumarylacetoacetate conversion ﾁ¨ succinylacetone
 Accumulated fumarylacetoacetate succinylacetone ⇒ toxic

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  Treatment strategies:
 Diet restriction of phyenylalanine, tyrosine, methionine
 Liver transplantation
 Metabolic inhibition of proximal pathway of tyrosine metabolism =
block formation of fumarylacetoacetate and succinylacetone
 Very rare disorder
 Clinical symptoms: eye and skin lesions, mental retardation
 Absence of tyrosine aminotransferase – enzyme involved in
transamination of tyrosine

Alkaptonuria
 Autosomal recessive disorder
 Rare
 Characterised by homogentisic aciduria, arthritis and ochronosis
 Hallmark of this disease: urine that is left standing becomes black
 Defect in tyrosine catabolic pathway
 Due to a deficiency of hepatic enzyme homogentisic acid oxidase
 Homogentisic acid (HGA) ↑↑ in blood and tissues and is passed in the
urine
 Upon contact with air, HGA oxidised ﾁ¨ pigmentlike polymeric material
alkapton = black colour of urine
 Earliest sign of disorder: when diapers stain black
 Disease progression is slow
 Childhood – early adulthood: asymptomatic, progressive deposition of
alkapton into collagenous tissues
 Ochronosis: external signs of alkapton deposition = bluish black
discoloration
 Discoloration of sclerae and ear cartilage ⇒ indicative of widespread
staining of body cartilage
 Most affected: the hips, knees, intervertebral joints
 Clinical symptoms resemble rheumatoid arthritis
 The condition is compatible with a normal life span, despite tendency for
arthritis later in life
 Homogentisic acid is a reducing substance and reacts with Clinitest tablets
 Currently no treatment

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