with Abnormal Urinary Constituents,

CHAPTER 9: Classified by Functional Defect

Overflow Inherited Meta

URINE bolic
Renal

SCREENING •Phenylketonuri
a
• Infantile •
tyrosinemia Hartn
• Tyrosinemia •Melanuria up
FOR • Alkaptonuria • Indicanuria diseas
• Maple syrup • 5- e
METABOLIC syndrome Hydroxyindo •
• Organic leacetic Cystin

DISORDERS acidemias
• Cystinosis
• Porphyria uria

• Porphyria
OVERFLOW •mucopolysacch
aridoses
 Results from the disruption of a normal • Galactosemia
metabolic pathway that causes increased • Lesch-Nyhan
plasma concentrations of the non- disease
metabolized substances

 Inherited lack of specific enzyme for NEWBORN SCREENING TEST

protein fat and carbohydrate metabolism –
 Screening of newborns has increased to
inborn error of metabolism
include more sensitive detection methods
RENAL and ever-increasing levels of state-mandated

 Caused by malfunctions in the tubular tests for Inborn Errors of Metabolism.

reabsorption mechanism  Many of these disorders cause the buildup

Major disorders of Protein and of unmetabolized toxic food ingredients, it is

Carbohydrates Metabolism Associated

important that the defects be detected early Disorder Amin rs
in life. s o
Acid
Heel Stick Blood
Disor
 Performed before infant leaves the ders
hospital. •Phenylk • • •
 Done 24rs after birth to within 3 days. etonuria Mapl Indicanuria Cyston
• e •5Hydroxyi uria
TESTS IN NEWBORN SCREENING:
Tyrosylu syrup ndoleacetic •
 Congenital hypothyroidism ria urine Acid Cystino

 Congenital hyperplasia •Alkapto diseas sis

nuria e •
 G6PD deficiency
• •Orga Homoc
 Galactosemia Melanuri nic ystinuri
a acade a
 Phenyllketonuria
mias
Tandem Mass Spectropotemetry
(MS/MS)

 Capable of screening the infant blood PHENYLALANINE-TYROSINE

sample for specific substances associated DISORDERS
with particular Inborn Errors of Metabolism.
 PHENYLKETONURIA (PKU)
 most well-known aminoacidurias
 failure to inherit the gene for
phenylalanine hydroxylase
production
 occur in 1 or every 10,000 to 20,000
births
AMINO ACID DISORDERS
 can lead to severe mental retardation
Phenylal Branc Trytophan Cystine  first detected in Norway by Ivan
anine- hed– Disorders Disorde Folling in 1934
Tyrosine Chain
  urine characteristics of Guthrie Test
peculiar―mousy odor (due to
Positive result of Phenylalanin-Tyrosine
increase amount of keto acids and
phenylpyruvate) Disorder is the growth of Bacillus subtilis.

 can be detected as early as 4 hours 2. Ferric Chloride Tube Test

after birth
- urine test for phenylpyruvic acid
 screening test for urine PKU if blood
level is elevated, it may take 2-6 Procedure:
weeks
a. Place 1mL of urine in a tube.
 phenyl alanine, a major constituent
b. Slowly add five drops of 10% ferric
of milk so dietary restriction is
chloride
recommended
 Many products that contain large c. Observe color for a permanent blue-green
amounts of phenylalanine, such as color
aspartame, now features warning
 TYROSYLURIA
labels for people with PKU.
TYPE 1 – caused by the deficiency of the
PKU Laboratory Tests
enzyme fumarylacetoacetate hydrolase
1. Guthrie Test or Guthrie’s Microbial (FAH)
Inhibition Assay
TYPE 2 – caused by lack of the enzyme
Procedure: tyrosine aminotranferase

a. Heel stick blood is absorbed into filter TYPE 3 – caused by lack of the enzyme p-
paper disks hydroxyphenylpyruvic acid dioxygenase

b. The blood impregnated disks are then TYROSYLURIA Laboratory Tests:

placed on culture media and streaked with
1. Ferric Chloride Test
organism of Bacillus subtilis.
(+): green color that fades rapidly
c. If increased phenylalanine levels present,
the phenylalanine will counteract the action 2. Nitroso-naphthol Test
beta-2-thienyalanine (the inhibitor of
- Recommended screening test for tyrosine
Bacillus subtilis)
in urine
- Also a non-specific test cause it reacts with - There is a high percentage of persons with
compounds other than proteins and it’s alkaptonuria develop liver and cardiac
metabolites disorders.

(+): Orange red color Manifestation in newborn:

Nitroso-napthol Test Procedure: 1. Brown/black stained cloth diapers

1. Place five drops of urine in a tube. 2. Reddish-stained in disposable diapers

2. Add 1mL of 2.63 nitric acid in Adult:

3. Add one drop of 21.5% sodium nitrite. 1. Brown pigment deposits in body tissues,
usually in the ears.
4. Add 0.1mL 1-nitro-2-naphthol
2. Deposits in the cartilage which lead to
5. Mix
arthritis
6. Wait for 5 minutes
ALKAPTONURIA Laboratory Tests:
7. Observe color
1. Ferric chloride test
 ALKAPTONURIA
(+): transient deep color
- Failure to inherit the gene for homogentisic
2. Clinitest
acid oxidase (without this enzyme, the
pheylalanine-tyrosine pathway cannot (+): yellow precipitate
proceed to completion, and homogentisic
3. Alkali Test
acid accumulates in the blood, tissues, and
(+): urine color becomes darker (upon
urine.
addition of alkali to a freshly voided urine
- was one of the six original inborn errors of
Interference: ascorbic acid
metabolism described by Garrod
4. Benedict’s Test
- Termed as “alkali lover” or alkaptonuria
because it darkens after becoming alkaline (+): yellow precipitate
from standing at room temperature.
5. Homogentisic Acid Taste
Reagent: Silver nitrate and Ammonium  5,6-dihydroxyindole = a colorless
Hydroxide precursor of melanin, which oxides to
melanogen and then to melanin producing
(+): black urine
the characteristics dark urine
Procedure:
MELANURIA Laboratory Test:
1. Place 4mL of 3% silver nitrate in a tube.
1. Ferric Chloride Tube Test
2. Add 0.5mL of urine.
(+): gray or black precipitate
3. Mix
2. Sodium Nitroprusside Test
4. Add 10% ammonium hydroxide
• Screening test for melanin
5. Observe for color black.
(+): Red color
6. Spectrophotometric Method
Interference: Acetone and Creatinine
- A quantitative test
BRANCHED-CHAIN AMINO ACID
Specimen: Blood or urine DISORDERS
It is very important to differentiate the  MAPLE SYRUP URINE
presence of homogentisic acid and melanin DISEASE
when urine is seen that has turned black
 An inborn error of metabolism, inherited
upon standing.
as an autosomal recessive trait
 MELANURIA
 Accumulation of Leucine, Isoleucine and
Melanin – the pigment responsible for the Valine in blood and urine
dark color of hair, skin and eyes.
MANIFESTATION:
 Increase melanin in urine which results to
1. Failure to thrive (newborn-after 1 week of
darkening of urine after exposure to air
birth)
 Albinism = deficiency of melanin
2. Maple syrup urine color
production
MAPLE SYRUP URINE DISEASE
 Melanocytes = melanin-producing cells
Laboratory Test:
1.) 2,4 – Dinitrophenylhydrazine (DNPH)  Accumulation of isovalerylglycine

 Most frequently performed screening test  Due to deficiency of isovalery-coenzyme

for keto acids A in the leucine body

 Not specific for MSUD  Has no urine screening test

NOTE: all specimens with a positive reagent  Propionic and Methylmalonic Acidimea
strip test result for ketones produce a
 Errors in metabolic pathway converting
positive DNPH result
isoleucine, valine, theonine and methionine
Procedure: to succinyl coenzyme A

1. Place 1mL of urine in a tube Propionic acid – immediate precursor to

methylmalonic acid
2. Add 10 drops of 0.2% 2,4-DNPH
 The presence of isovalericpropionic and
3. Wait for 10 minutes
methylmionic acemias can be detected by
4. Observe for yellow or white precipitate new born screening programs by using

 ORGANIC ACIDEMIAS Thandem spectrophotometry

General manifestations: ORGANIC ACIDEMIAS Laboratory Test

(Methylmalonic Aciduria)
a. Vomiting accompanied by metabolic
acidosis Para/p-Nitroaniline Test

b. Hypoglycemia 1. Place one drop of urine in a tube.

c. Ketonuria 2. Add 15 drops of 0.1% p-nitroaniline in

0.16 M HCl
d. Increased serum ammonia
3. Add five drops of 0.5% sodium nitrite
Three most frequently encountered
disorders: 4. Mix

 Isovaleric Acidemia 5. Add 1mL of 1M sodium acetate buffer at

pH 4.3
 Urine characteristics of ―sweaty feet
odor 6. Boil for 1 minute
7. Add five drops of 8N NaOH  2nd metabolic pathway pf tryptophan

8. Observe for emerald green color (+)  For production of serotonin used in the
stimulation of smooth muscles degradation
TRYPTOPHAN DISORDERS
product of serotonin is 5-HIAA which is
Metabolism of Tryptophan: excreted in urine.
Tryptophan enters the intestine either by: Serotonin = produced from tryptophan by
a. Reabsorbed for use by the body for the argentaffin cells in the intestine and in
production of proteins carried through the body primarily by the
platelets.
b. Converted to indole by intestinal bacteria
then excreted in the feces NOTE: normal value of 5 HIIA, 2-8mg for
24 hours.
 INDICANURIA
>8mg indicates argentaffin tumor
 Can be due to intestinal disorders
(obstruction, presence of abnormal bacteria 5-HYDROXYINDOLEACETIC ACID

and malabsorption syndrome) Laboratory Test:

 Increased urinary indicant Nitroso-naphthol Test/Silver

Nitroprusside Test
 Hartnup Disease
(+): purple to black color
 Also known as blue diaper Syndrome
Specimen of choice:
(blue staining on infant’s diaper)
a. Random urine
 a rare inherited disorder than can exhibit
Indicanuria b. First morning urine

 Affects the intestinal reabsorption of * False-negative results can occur based

tryptophan and renal Tubular reabsorption on the specimen concentration and also
of amino acids resulting in a generalized because 5-HIAA may not be produced at
aminoaciduria called Fanconi Syndrome a constant rate throughout the day. If a
24-hour sample is used, it must be
 5-HYDROXYINDOACETIC
preserved with hydrochloric or boric
ACID
acid.
* For 24 sample, Patients must be given 2. Only lysine and cystine are not
explicit dietary instructions before reabsorbed
collecting any sample to be tested for 5-
CYSTINURIA Laboratory Test
HIAA, because serotonin is a major
constituent of foods such as bananas, Cyanide-Nitroprusside Test

pineapples, and tomatoes. Procedure:

Interference: phenothiazines and Acetanilids 1. Place 3mL of urine in a tube

Patients should be directed to withhold 2. Add 2mL sodium cyanide

medications for 72 hours before specimen
3. Wait for 10 minutes
collection.
4. Add five drops 5% sodium nitroprusside
CYSTINE DISORDERS
5. Observe for red-purple color
 Inherited disorders that have
characteristics of sulfur odor of urine with Results:
cystine metabolism disorder
(+): red purple color
 CYSTINURIA (False +): if there presence of ketones and
 Elevated level of the amino acid cystine hemocystine
in the urine  CYSTINOSIS
 The presence of increased urinary cystine
 An inborn error of metabolism where
is not due to a defect in the metabolism of
cystine crystals deposit in many areas if the
cystine but rather than,
body (Cornea, bone marrow, lymph nodes,
 Inability of the renal tubules to reabsorb internal
cystine filtered by the glomerulus
organs).
 It forms renal calculi
 major defect in the renal tubular
Two modes of inheritance: reabsorption mechanism (Fanconi
syndrome) also occurs wo general
1. Disorder in the reabsorption of 4 amino
categories:
acids: (COLA) cystine, ornithine, lysine and
arginine a. Nephropatic
  Infantile Silver Nitroprusside Test
 there is rapid progression to renal
 The use of silver nitroprusside test in
failure
place of sodium cyanide reduces
 Routine laboratory findings in
homocysteine to it nitroprusside reactive
infantile nephropathic cystinosis
form but does not reduce
include polyuria, generalized
aminoaciduria, positive Clinitest cysteine

results for reducing substances, and  Fresh urine must be used when testing
lack of urinary concentration.
Procedure:
 Late-onset
 a gradual progression to total renal 1. Place 1mL of urine in a tube
failure
2. Add 2 drops concentrated ammonium
b. Non-nephropatic
hydroxide
 relatively benign but may cause some
3. Add 0.5mL 5% silver nitrate
ocular
4. Wait for 10mins
disorders.
5. Add 5 drops nitroprusside
 HOMOCYSTINURIA
6. Observe for red-purple color (positive
 Defect in the metabolism of methionine result)
leading to increased homocysteine
PORPHYRIN DISORDERS
MANIFESTATION:
Porphyrin – are the intermediate compounds
a. Failure to thrive in the production of heme
b. Cataracts Primary porphyrin
c. Mental retardation a. Urophorphyrin
d. Thromboembolic problems b. Coproporphyrin
e. Death c. Protoporphyrin
HOMOCYSTINURIA Laboratory Test
d. Porphyrin precursors – aminolevulinic  PORPHYNURIA
acid (ALA) and porphobolinogen (most
- Red or port wine color of urine after
soluble and readily appear
exposure to air
 Fecal analysis has usually been performed
Congenital porphyria – red discoloration of
for the detection of coproporphyrin and
an
protoporphyrin.
infant’s diaper
 To avoid false positive interference bile is
a more acceptable specimen. TWO SCREENING TESTS FOR
PORPHYNURIA:
 Whole blood is recommended for the
presence of free erythrocyte protoporphyrin 1. Erlich reaction – used only for the

as a screening test for lead poisoning. detection of ALA and porphobilinogen

 PORPHYRIAS 2. Fluorescent technique – used for the

porphyrins. It is under ultra violet light
- Either inherited or acquired from
erythrocytic and hepatic malfunctions or (+): violet, pink or red flourescence

exposure to toxic (-): faint blue fluorescence

agent MUCOPOLYSACCHARIDE

- Inherited – either neurologic or psychiatric DISORDERS

or cutaneous photosensitivity or a - Or glycosaminoglycans are inherited

combination of disorders which results in accumulation of

both. the incompletely metabolized

polysaccharide portions in the lysosomes of
Common causes of Acquired Porphyrias:
connective tissue cells and their increase
a. Lead poisoning excretion in urine

b. Excessive alcohol exposure Best known Mucopolysaccharides:

c. Iron deficiency  Hurler Syndrome –

mucopolysaccharides
d. Chronic liver disease

e. renal disease
accumulate in the cornea; skeletal structure 3. Mucopolysaccharide (MPS) Paper Test
abnormality and severe mental retardation
Procedure:
 Hunter Syndrome – skeletal structure
1. Dip filter paper into 0.59% azure A dye in
abnormality and severe mental retardation.
2% acetic acid
An inherited as sex-link recessive and rarely
seen in 2. Dry

females. 3. Add one drop of urine to paper

 Sanfilippo Syndrome – mental 4. Wash with 1mL acetic acid + 200mL

retardation only methanol diluted to a liter

Hurler and Hunter Syndrome are both 5. Observe for blue colof

fatal during childhood if not treated. PURINE DISORDERS

Bone marrow transplant and Gene  Lesch-Nyhan Disease

replacement therapy
- Inherited as a sex-linked recessive result in
- Most promising treatments for these massive excretion of urinary uric acid
disorders crystals

MUCOPOLYSACCHARIDE Screening - Caused by the failure to inherit gene to

Tests: produce hypoxanthine guanine

1. Acid albumin phosphoribosyl transferase (responsible for

the accumulation of uric acid throughout the
2. Cetytrimenthylammonium Bromide
body)
(CTAB) Test
- ―Orange Sand in diaper
Procedure:
CARBOHYDRATES DISORDERS
1. Place 5mL of urine in a tube
 Melituria
2. Add 1mL of 5% CTAB in citrate buffer
- presence of increased urinary sugar due to
3. Read turbidity in 5 minutes
an inherited disorder.
(+): white turbidity
- The majority of melituria caused no 1. Place 5mL of urine in a tube
disturbance to body metabolism except for
2. Add 5mL of 25% HCl
galactosuria
3. Boil for 5 minutes
 Galactosuria
4. Boil for 10 seconds
- Inability to properly metabolize galactose
5. Observe for a red precipitate
to glucose

- Caused by a deficiency in any of the 3

enzymes:

a. Galactose-1-phosphate uridyl
transferase (GALT)

- Deficiency of this causes the most severe

possible fetal symptoms associated with
Galactosemia

b. Galactokinase

c. UDP-galactose-4-epimerase

New new born screening test currently test

for the presence of GALT deficiency

Lactosuria – seen during pregnancy

Fructosuria – associated with parental

feeding

Pentosuria – ingestion of large amounts of

fruits

Fructose screening test

Resorcinol Test

Procedure: