CHAPTER 8: URINE SCREENING FOR METABOLIC DISORDERS
- Include phenylketonuria (PKU), tyrosyluria, alkaptonuria,
Abnormal Conditions Detected in the Routine Urinalysis
Overflow Versus Renal Disorders
The appearance of abnormal metabolic substances in the urine
can be caused by a variety of disorders that can generally be
grouped into two categories:
 Overflow
- result from disruption of a normal metabolic pathway that
causes increased plasma concentrations of the
nonmetabolized substances.
- Overrides Tm or is not normally reabsorbed
- Abnormal accumulations: caused by malfunctions in the
tubular reabsorption mechanism.
- Inborn Error of Metabolism: failure to inherit the gene to
produce a particular enzyme. (protein,fat, or carbohydrate
metabolism)
 Renal
- Functional defects
Disorders Classified by Defect
Newborn Screening
- Current state-mandated screening for as many as 30
or more metabolic disorders
- Many disorders cause the buildup of unmetabolized
toxic food ingredients (should be detected early in
life). Levels are elevated more rapidly in blood than
urine.
- Urine tests are primarily for follow-up
- Heel puncture or Heel stick blood tests are used for
initial testing
 Performed before infant leaves hospital
 Metabolites appear first in the blood
- Analyze by tandem mass spectrophotometry and…
- MS/MS - capable of screening the infant blood
sample for specific substances associated with
particular IEMs.
 Gene testing is being worked on
Amino Acid Disorders (Aminoacidurias)
melanuria,maple syrup urine disease, organic acidemias,
indicanuria, cystinuria, and cystinosis.
-
 Phenylalanine-Tyrosine Disorders
- Major inherited disorders: PKU, tyrosyluria, and
alkaptonuria.
- Metabolic defects cause overproduction of melanin.
1. Phenylketonuria
- The most well known of the aminoacidurias
- 1 in 10,000 births to 20,000 births
- Results in severe mental retardation
- First identified in Norway by Ivan Følling in 1934,
Urine: a peculiar mousy odor (increased amounts of
the keto acids, including phenylpyruvate)
- occurs when the normal conversion of phenylalanine
to tyrosine is disrupted (tyrosine decreases with its
pigmentation metabolite melanin).
- caused by failure to inherit the gene to produce the
enzyme phenylalanine hydroxylase
- Autosomal-recessive trait; heterozygotes normal
- Once discovered: Eliminate phenylalanine from diet
(milk) since it damages to child’s mental capacity and
avoid ↑ phenylalanine foods (aspartame).
- increased blood levels of phenylalanine must occur
before urinary excretion of phenylpyruvic acid (2 to 6
weeks).
- Phenylalanine can be detected as early as 4 hours
after birth and, if the normal results 4mg/dl is
lowered 2 mg/dL, the presence of PKU should be
detected.
 Guthrie blood test
 Media containing beta-2-thienylalaline, an inhibitor of
Bacillus subtilis, is streaked with the bacteria; blood-
impregnated discs placed on the agar; phenylalanine
counteracts the inhibitor.
 Urine test:
- Ferric chloride tube test result to blue-green color
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2. Tyrosyluria/Tyrosinemia
- accumulation of excess tyrosine in the plasma
(tyrosinemia) producing urinary overflow.
- the urine may contain its degradation products, p-
hydroxyphenylpyruvic acid and p-hydroxyphenyllactic
acid.
 Metabolic defects
- Premature transitory tyrosinemia (infants), which is
caused by underdevelopment of the liver function
- Acquired severe liver disease also produces
tyrosyluria resembling transitory tyrosinemia.
- Results:
o rarely seen tyrosine and leucine crystals
 Hereditary defects
- enzymes required in the metabolic pathway are not
produced resulting to the liver and renal tubular
disease that produces aminoaciduria.
- Type 1: enzyme deficiency is fumarylacetoacetate
hydrolase (FAH); renal tubular disease and liver
failure in infants
- Type 2: enzyme deficiency is tyrosine
aminotransferase; corneal erosion and lesions on
palms, fingers, and soles of the feet believed to be
caused by crystallization of tyrosine in the cells.
- Type 3: enzyme deficiency is p-hydroxyphenylpyruvic
acid dioxygenase; mental retardation if no dietary
restrictions of phenylalanine and tyrosine.
- All types produces tyrosylemia and tyrosyluria.
 Urine tests
- Ferric chloride produces a transient green color
- Nitroso-naphthol test produces an orange-red color
 Branched Chain Amino Acid Disorders
3. Melanuria
 Second pathway for tyrosine
- Melanin, thyroxine, epinephrine, protein, and
tyrosine sulfate.
 Melanin
- Pigment for dark hair, skin and eyes
- Deficiency causes albinism
- Increased urinary melanin = darkens the urine (after
the urine is exposed to air or oxidation of melanogen)
- Elevated urinary melanin = indicates proliferation of
the normal melanin-producing cells (melanocytes),
producing a “malignant melanoma.”
- 5,6-dihydroxyindole - a colorless precursor of melanin
which oxidizes to melanogen and then to melanin,
producing the characteristic dark urine. (Secreted by
the Tumors)
 Urine tests
- Ferric chloride produces a Black precipitate
- Sodium nitroprusside test produces a Red color
4. Alkaptonuria
- Enzyme deficiency is homogentisic acid oxidase
- homogentisic acid accumulates in the blood, tissues,
and urine.
- one of the six original inborn errors of metabolism
described by Garrod in 1902
- a metabolic defect that is the third major defect in
the phenylalanine-tyrosine pathway
- the urine darkens after becoming alkaline from
standing at room temperature
- Black alkaline urine, possible black-stained diapers
- Manifests later in life with brown pigment deposits in
tissues (ears):
1. Deposits in the cartilage eventually lead to
arthritis.
2. Develops liver and cardiac disorders
- Another screening test:
Add alkali to freshly voided urine and to observe for
darkening of the color; but ascorbic acid interferes
- Paper and thin layer chromatography procedures are
available.
 Urine tests
- Ferric chloride produces a transient deep blue color
- Clinitest produces yellow precipitate
(indicating the presence of a reducing substance.)
- Silver nitrate and ammonium hydroxide test
produces black color
- differ from other amino acids by having a methyl
group that branches from the main aliphatic carbon
chain
- Two major groups:
1. Maple syrup urine disease: accumulation of one
or more of the early amino acid degradation
products
2. Organic acidemias: accumulation of organic acids
produced further down in the amino acid
metabolic pathway.
- A significant laboratory finding: presence of ketonuria
1. MSUD
- Inborn error of metabolism, autosomal-recessive trait
- Amino acids involved are:
= leucine, isoleucine, and valine
- metabolic pathway begins by the transamination of
the three amino acids in the liver to the keto acids:
1. a –ketoisovaleric
2. a -ketoisocaproic,
3. a -keto-b -methylvaleric.
- Enzymes are necessary to produce oxidative
decarboxylation of these keto acids, failure to inherit
results in the accumulation of keto acids in the blood
and urine.
- Newborns with MSUD begin to exhibit clinical
symptoms: 1-week failure to thrive
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 - Result in Urine: strong odor of maple syrup, and thick,
dark appearance
- detected by the 11th day, dietary regulation and
careful monitoring of urinary keto acid concentrations
 Screening test:
2,4-dinitrophenylhydrazine (DNPH) produces yellow
precipitate turbidity
2. Organic Acidemias
- Symptoms: early severe illness, vomiting, metabolic
Acidosis, hypoglycemia, ketonuria, and increased
serum ammonia.
- Most common disorders are:
1. Isovaleric acidemia: “sweaty feet odor”
o caused by the accumulation of
isovalerylglycine due to a deficiency of
isovaleryl coenzyme A in the leucine
pathway.
2– 3. Propionic and methylmalonic acidemia:
o no conversion of valine, threonine,
methylmalonate to succinyl coenzyme A.
o Propionic acid is the immediate precursor.
 Urine tests
- p-Nitroaniline Test produces Emerald green color
- Procedures:
1. Place 1 drop of urine in a tube
2. Add 15 drops of 0.1% p-nitroaniline in 0.16 M HCl
3. Add 5 drops of 0.5% sodium nitrite
4. Mix
5. Add 1 mL of 1 M sodium acetate buffer at pH 4.3
6. Boil for 1 min
7. Add 5 drops of 8 N NaOH
8. Observe for emerald green color
 Tryptophan Disorders
- increased urinary excretion of the metabolites indican
and 5-hydroxyindoleacetic acid (5-HIAA).
 Cystine Disorders
1. Indicanuria
- most of the tryptophan that enters the intestine is
either for protein production or is converted to indole
by intestinal bacteria and excreted in the feces.
- Some Intestinal disorders with increased amounts of
tryptophan that are converted to indole:
1. obstruction;
2. presence of abnormal bacteria;
3. malabsorption syndromes;
4. Hartnup disease
- excess indole is reabsorbed from the intestine into
the bloodstream and circulated to the liver, where it
is converted to indican and then excreted in the
urine.
- Urine Results: colorless until oxidized
to the dye indigo blue by exposure to air.
 Hartnup disease: blue diaper syndrome
- affects the intestinal reabsorption of tryptophan and
the renal tubular reabsorption of other amino acids.
- results in aminoaciduria (Fanconi syndrome)
 Requires dietary supplements (niacin)
- Urine Results: Blue or Violet color with ferric chloride
that can be extracted into chloroform
2. 5-Hydroxyindoleacetic Acid (5-HIAA)
- Tryptophan produces serotonin in the second
metabolic pathway.
- Serotonin is used for the stimulation of smooth
muscles and produced by the argentaffin cells in the
intestine. It is carried through the body primarily by
the platelets.
- Excess serotonin is excreted in the urine as 5-HIAA
- Carcinoid tumors involving the argentaffin
(enterochromaffin) cells: ↑ 5-HIAA in urine due to
excess serotonin produced.
- Normal daily excretion of 5-HIAA: 2 to 8 mg/day
- Argentaffin cell tumors: >25 mg/day
 Urine test:
- Adding “Nitrous acid” and “1-nitroso-2-naphthol” to
5-HIAA produces purple to black color
- can be performed on a random or first morning
specimen
- False-negative results: based on the specimen
concentration and inconsistent production of 5-HIAA.
- 24-hour sample must be preserved with
= hydrochloric or boric acid.
- Patient instructions:
 No bananas, pineapples, tomatoes (serotonin
is a major constituent)
 No phenothiazines, and acetanilids for 72 hours
- A plasma method using high-performance liquid
chromatography with fluorescence detection is also
available.
o Two distinct disorders of cystine metabolism
exhibit renal manifestations.
o Both have noticeable odor of sulfur
o Difference:
1. Cystinuria - a defect in the renal tubular
transport of amino acids
2. Cystinosis - inborn error of metabolism
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 - Gives a positive result with the cyanide-nitroprusside
1. Cystinuria test but requires an additional screening test. Which
- marked by elevated amounts of the amino acid is this test (silver-nitroprusside) where only
cystine in the urine. (due to inability of the renal homocystine will react.
tubules to reabsorb cystine filtered by the - The use of silver nitrate in place of sodium cyanide
glomerulus) reduces homocystine to its nitroprusside-reactive
- Inherited disorder affecting renal reabsorption form but does not reduce cystine.
- Two modes of inheritance: - Fresh urine should be used when testing for
1. only cystine and lysine are not reabsorbed homocystine.
2. cystine, lysine, arginine, and ornithine are not
reabsorbed
- may form renal calculi but more common (65%) on
people with 4 amino acids inheritance.
- Cystine is the least soluble accounting for cystine
crystals in the sediment of concentrated or first
morning specimens. It is the only amino acid found
during the analysis of calculi

 Screening test:  Porphyrin Disorders

cyanide-nitroprusside test - are the intermediate compounds in the production of
 Sodium cyanide reduces cystine, and nitroprusside heme.
produces a red-purple color if excess cystine is
present
 False-positives: ketonuria, homocystinuria

Cystinosis
 Inborn error of metabolism
 Three variations:
A. Nephropathic
1. Infantile nephropathic cystinosis - rapid
progression to renal failure.
o Results: polyuria, generalized
aminoaciduria, positive Clinitest results
for reducing substances, and lack of
urinary concentration.
2. late-onset nephropathic cystinosis - a gradual
progression to total renal failure.
B. Non-nephropathic
- benign, some ocular problems
 Defect in lysosomal membranes prevents release of
cystine into cytoplasm for metabolism = crystalline
cystine deposits in body
 Deposits: cornea, bone marrow, lymph nodes, organs
 Renal tubules are affected by deposits causing
Fanconi syndrome, which is not inherited
 Renal transplants and cystine-depleting medications
- Primary porphyrins: uroporphyrin, coproporphyrin,
Homocystinuria protoprophyrin
- Defect in metabolism of methionine, producing - Precursors: α-aminolevulinic acid (ALA) and
increased methionine in body porphobilinogen
- The increased homocystine can result: Failure to - Detection of pathway disruptions in urine, blood,bile/feces
thrive, cataracts, mental retardation, thromboemboli, - The solubility of the porphyrin compounds
and death. 1. Urine: ALA, porphobilinogen, urobilinogen (Three
Most soluble), coproporphyrin (least soluble)
 Screening test: 2. Feces/bile: coproporphyrin and protoporphyrin
- Silver Nitroprusside Test produces a red-purple color Note: to avoid false-positive interference, bile is a
more acceptable specimen.
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 3. Whole Blood: presence of free erythrocyte
protoporphyrin (a screening test for lead poisoning.)
- Disorders of porphyrin metabolism are collectively termed
“porphyrias” from erythrocytic and hepatic malfunctions
or exposure to toxic agents
A. Inherited: gene in metabolic pathway is missing
- Classified by clinical symptoms as neurologic/psychiatric,
cutaneous/photosensitivity, or both
B. Acquired (more common): lead poisoning, alcoholism,
iron deficiency, chronic liver and renal disease
- Porphyrinuria indicator: observation of a red or port wine
color to the urine after exposure to air.
- Erythropoietic porphyrias: when the port wine urine color
is more prevalent; staining of the teeth may also occur.
- Congenital porphyria: a red discoloration of an infant’s
diaper.
 Screening test:
1. Ehrlich reaction: only for ALA and porphobilinogen
 Convert ALA to porphobilinogen by adding acetyl
acetone
A. Watson-Schwartz test – differentiation between
the presence of urobilinogen and
porphobilinogen.
B. Hoesch test - testing for the presence of
porphobilinogen when patients exhibit
symptoms of an acute attack.
o A negative test result is obtained in the presence of
lead poisoning unless ALA is first converted to
porphobilinogen.
2. Fluorescence under ultraviolet light (550- to 600-nm) –
- used for other porphyrins
- extraction into glacial acetic acid and ethyl acetate
- The solvent layer is then examined.
- Negative reaction: faint blue fluorescence.
- Positive reaction: fluoresce violet, pink, or red, depending
on the concentration of porphyrins.
- If the presence of interfering substances is suspected:
1. the organic layer can be removed to a separate tube
2. Add 0.5 mL of hydrochloric acid to the tube
Note: Only porphyrins are extracted into the acid layer,
which then produces a bright orange-red fluorescence.
- does not distinguish among uroporphyrin, coproporphyrin,
and protoporphyrin, but rules out porphobilinogen and
ALA.
- Increased protoporphyrin is best measured in whole
blood.
Mucopolysaccharide Disorders
- Mucopolysaccharides, or glycosaminoglycan - a group of
large compounds located primarily in the connective
tissue.
- Inherited disorders in the metabolism of preventing
complete breakdown of the polysaccharide portion of the
compounds.
- Results: accumulation of the incompletely metabolized
polysaccharide portions in the lysosomes of the connective
tissue cells and their increased excretion in the urine.
- Substances found in urine: dermatan sulfate, keratan
sulfate, and heparin sulfate
- Mucopolysaccharidoses Types:
 Hurler syndrome: abnormal skeletal structure, severe
mental retardation, and corneal eye damage
 Hunter syndrome: abnormal skeletal structure,
severe mental retardation, inherited as a sex-linked
recessive, rare in females
 Sanfilippo syndrome: mental retardation
- Bone marrow transplantation and gene therapy for
treatment
 Screening test:
1. Acid-albumin and cetyltrimethylammonium bromide
turbidity test (CTAB)
 Thick, white turbidity forms when reagents are added
 Turbidity is usually graded on a scale of 0 to 4 after
30 minutes with acid-albumin and after 5 minutes
with CTAB.
2. Metachromatic staining spot test
 Positive urine produces a blue color that cannot be
washed away with dilute acidified methanol
 Mucopolysaccharide Paper Test
1. Dip filter paper into 0.59% azure A dye in 2%
acetic acid
2. Dry
3. Add 1 drop of urine to paper
4. Wash with 1 mL acetic acid + 200 mL methanol
diluted to a liter
5. Observe for blue color
 Purine Disorders
1. Lesch-Nyhan disease
- Inherited as a sex-linked recessive results in
massive excretion of urinary uric acid crystals.
- Failure to inherit the gene to produce the enzyme
hypoxanthine guanine phosphoribosyltransferase is
responsible for the accumulation of uric acid.
- Results: Motor defects, mental retardation, self-
destruction, gout and renal calculi.
- Normal development 6–8 months
- First sign” uric acid crystals resembling orange sand
in diapers
 Carbohydrate Disorders
- An inherited disorder: presence of increased urinary
sugar (melituria
- No problems except for galactosuria; indicating the
inability to properly metabolize galactose to glucose.
- caused by a deficiency in any of three enzymes,
galactose-1-phosphate uridyl transferase (GALT),
galactokinase, and UDP-galactose-4-epimerase.
- Failure to thrive, severe mental retardation, cataracts,
liver disorders
1. Lactosuria
- Pregnancy and lactation
2. Fructosuria
- Parenteral feeding
- Resorcinol screening test
3. Pentosuria
- Ingestion of large amounts of fruit
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