Download urine screening for metabolic disorders - 36-454-f10

URINE SCREENING FOR
METABOLIC DISORDERS
CHAPTER 8
Copyright © 2014. F.A. Davis Company
Learning Objectives
Upon completing this chapter, the reader will be able to
1. Explain abnormal accumulation of metabolites in the urine in terms of
overflow and renal disorders.
2. Discuss the importance of and the MS/MS testing methods for
newborn screening.
3. Name the metabolic defect in phenylketonuria, and describe the
clinical manifestations it produces.
4. State three causes of tyrosyluria.
5. Name the abnormal urinary substance present in alkaptonuria, and
explain how its presence may be suspected.
6. Discuss the appearance and significance of urine that contains
melanin.
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Learning Objectives (cont’d)
7. Describe a basic laboratory observation that has relevance in
maple syrup urine disease.
8. Discuss the significance of ketonuria in a newborn.
9. Differentiate between the presence of urinary indican owing
to intestinal disorders and Hartnup disease.
10. State the significance of increased urinary 5hydroxyindoleacetic acid.
11. Differentiate between cystinuria and cystinosis, including the
differences found during analysis of the urine and the disease
processes.
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Learning Objectives (cont’d)
12. Describe the components in the heme synthesis pathway,
including the primary specimens used for their analysis, and
explain the cause and clinical significance of major porphyrias
and the appearance of porphyrins in urine.
13. Define mucopolysaccharides, and name three syndromes in
which they are involved.
14. State the significance of increased uric acid crystals in
newborns’ urine.
15. Explain the reason for performing tests for urinary-reducing
substances on all newborns.
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Overflow Versus Renal Disorders
• Overflow
– Disruption of a normal metabolic pathway
– Increased plasma concentrations of the nonmetabolized
substances
– Overrides reabsorption ability of renal tubules
– Inherited lack of specific enzyme for protein, fat, or
carbohydrate metabolism—inborn error of metabolism
• Renal
– Malfunctions in the tubular reabsorption mechanism
Copyright © 2014. F.A. Davis Company
Disorders Classified by Defect
Overflow Inherited
Metabolic
Renal
Phenylketonuria
Tyrosinemia
Infantile tyrosinemia
Melanuria
Hartnup disease
Cystinuria
Alkaptonuria
Indicanuria
Maple syrup urine disease
5-Hydroxyindoleacetic acid
Organic acidemias
Porphyria
Cystinosis
Porphyria
Mucopolysaccharidoses
Galactosemia
Lesch-Nyhan disease
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Phenylalanine/Tyrosine
Metabolic Pathway
• Current state-mandated screening for as many as 30 or
more inborn errors of metabolism (IEM)
• Urine tests are primarily for follow-up
• Disorders can cause abnormal urinalysis results
• Heel stick blood tests are used for testing
– Performed before infant leaves hospital
– But >24 h
– Metabolites appear first in the blood
• Analyze by tandem mass spectrophotometry, MS/MS
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Phenylalanine/Tyrosine
Metabolic Pathway
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Amino Acid Disorders
(Aminoacidurias)
• Phenylketonuria, tyrosyluria, alkaptonuria
• Phenylketonuria
– 1 in 10,000 to 20,000 births
– Autosomal recessive; heterozygotes normal
– Eliminate phenylalanine from diet (milk)
– Damage to child’s mental capacity
– Alternate pathways as child matures
– Avoid ↑ phenylalanine foods (aspartame)
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Amino Acid Disorders
(Aminoacidurias)(cont'd)
• Phenylalanine hydroxylase is missing
• Urine test
– Urine and 5% ferric chloride produces a permanent
green-blue color
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Tyrosyluria/Tyrosinemia
• Metabolic defects
– Premature transient tyrosinemia
• Underdevelopment of liver function
– Acquired severe liver disease
• Hereditary defects
– Type 1: enzyme deficiency is fumarylacetoacetate
acid hydrolase; renal tubular disease and liver failure
in infants
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Tyrosyluria/Tyrosinemia (cont’d)
• Hereditary defects
– Type 2: enzyme deficiency is tyrosine
aminotransferase; corneal erosion and lesions on
hands and feet
– Type 3: enzyme deficiency is phydroxyphenylpyruvate oxidase; mental retardation if
no dietary restrictions (milk)
• Screening tests
– Screening tests using MS/MS are available for
tyrosinemia types 1, 2, and 3
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Melanuria
• Second pathway for tyrosine
– Melanin, thyroxine, epinephrine, protein, and
tyrosine sulfate
• Melanin
– Pigment for dark hair, skin
– Defect causes albinism
– Increased production = malignant melanoma
– 5,6-dihydroxyindole
• Dark urine from oxidation of melanogen to melanin
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Alkaptonuria
• Enzyme deficiency is caused by a failure to inherit the
gene to produce the enzyme homogentisic acid oxidase
• Third major defect in the phenylalanine-tyrosine pathway
• Black alkaline urine, possible black-stained diapers
• Manifests later in life with brown pigment deposits in
tissues
• Urine: blue with ferric chloride, yellow precipitate with
Clinitest, black with silver nitrate and ammonium
hydroxide; quantitative tests available
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Branched Chain
Amino Acid Disorders
• Amino acids with a methyl group that branches
from the main aliphatic carbon chain
• Two groups
1. Maple syrup urine disease (MSUD); early
degradation products accumulate
2. Organic acidemias; accumulation of organic acids
further down in pathway
• Ketonuria in a newborn
Copyright © 2014. F.A. Davis Company
Maple Syrup Urine Disease
(MSUD)
•
•
•
•
Inborn error of metabolism, autosomal recessive
Amino acids involved are leucine, isoleucine, and valine
1-week failure to thrive is noticed
Urine: strong odor of maple syrup, and thick, dark
appearance
• Dietary regulation by day 11 shows good outcomes
• Positive urine ketones
• Screening test 2,4-dinitrophenylhydrazine produces yellow
precipitate turbidity
Copyright © 2014. F.A. Davis Company
2,4-Dinitrophenylhydrazine
(DNPH) Test
1.
2.
3.
4.
Place 1 mL of urine in a tube.
Add 10 drops of 0.2% 2,4-DNPH in 2N HCl.
Wait 10 minutes.
Observe for yellow or white precipitate.
Copyright © 2014. F.A. Davis Company
Organic Acidemias
• Early: severe vomiting, metabolic acidosis, hypoglycemia,
ketonuria
• Isovaleric, propionic, methylmalonic acidemias
• Isovaleric: “sweaty feet odor” from patient
– Deficiency of isovaleryl coenzyme A
• Propionic and methylmalonic: no conversion of valine,
threonine, methylmalonate to succinyl coenzyme A
• Isovaleric, propionic, and methylmalonic acidemias can be
detected by newborn screening programs using MS/MS
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Tryptophan Disorders
• Increased urinary
excretion of the
metabolites indican and
5-hydroxyindoleacetic
acid (5-HIAA)
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Indicanuria
• Tryptophan enters intestine, is reabsorbed or is
converted to indole by bacteria, and leaves in the
feces
• Intestinal disorders and Hartnup disease cause
increased tryptophan conversion to indole
• Increased indole reabsorbed, excreted by kidney
on its way to the liver
• Exposure of urine to air = indigo blue
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Indicanuria (cont’d)
• Hartnup disease: blue diaper syndrome
– Inherited disorder affects intestinal reabsorption of
indole and renal tubular reabsorption = Fanconi
syndrome
– Requires dietary supplements: niacin
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5-Hydroxyindoleacetic
Acid (5-HIAA)
• Tryptophan produces serotonin
• Serotonin from tryptophan is produced by the
intestinal argentaffin cells and is carried in the
body to the muscles by platelets
• Excess excreted in the urine as 5-HIAA
• Argentaffin (enterochromaffin) cell tumors = ↑
↑ 5-HIAA in urine from excess serotonin
produced
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5-Hydroxyindoleacetic
Acid (5-HIAA)(cont'd)
• Urine test
– Nitrous acid and 1-nitroso-2-naphthol produce purple to black
color
– Normal 2 to 8 mg/day, >25 mg/day in disease
– Can perform test on random specimens
– Patient instructions
• No bananas, pineapples, tomatoes, phenothiazines, and acetanilids for
72 hours
• 24-hour urine samples must be preserved with HCl or boric acid
Copyright © 2014. F.A. Davis Company
Cystine Disorders
• Two different disorders; both have noticeable odor
of sulfur
• Cystinuria
– Inherited disorder affecting renal reabsorption
– Two modes of inheritance: (1) only cystine and lysine are
not reabsorbed; (2) cystine, lysine, arginine, and ornithine
are not reabsorbed
– Increased calculi formation early in life for both modes
– Approximately 65% of the people in whom all four amino
acids are affected can be expected to produce calculi
early in life
Copyright © 2014. F.A. Davis Company
Cystine Disorders (cont’d)
• Cystine is the least soluble accounting for cystine
crystals
• Cystine is also the only amino acid found during the
analysis of calculi from these patients
• Urine screening test: cyanide-nitroprusside
– Na cyanide reduces cystine, and nitroprusside produces a
red-purple color if excess cystine is present
– False-positives: ketonuria, homocystinuria
Copyright © 2014. F.A. Davis Company
Cyanide-Nitroprusside Test
1.
2.
3.
4.
5.
Place 3 mL of urine in a tube.
Add 2 mL sodium cyanide.
Wait 10 minutes.
Add five drops 5% sodium nitroprusside.
Observe for red-purple color.
Copyright © 2014. F.A. Davis Company
Cystinosis
• Genuine IEM
• Ranging from a severe fatal disorder developed in infancy to a
benign form appearing in adulthood
• Two categories
– Nephropathic
• Infantile
• Later life
– Non-nephropathic
• Defect in lysosomal membranes prevents release of cystine
into cytoplasm for metabolism = crystalline cystine deposits in
body
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Cystinosis (cont’d)
• Deposits: cornea, bone marrow, lymph nodes, organs
• Renal tubules are affected by deposits causing Fanconi
syndrome, which is not inherited
• Infantile: rapid progression to renal failure
• Late-onset: gradual progression to renal failure
• Non-nephropathic: benign, some ocular problems
• Laboratory: aminoaciduria, reducing substances, cystine
crystals
• Renal transplants and cystine-depleting medications
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Homocystinuria
• Defect in metabolism of methionine, producing
increased methionine in body
• Failure to thrive, cataracts, mental retardation,
thromboemboli, death
• Requires changes in diet
• Additional screening with silver nitrate instead of
sodium cyanide
• Included in newborn screening programs performed
using MS/MS testing
Copyright © 2014. F.A. Davis Company
Silver Nitroprusside Test
1.
2.
3.
4.
5.
6.
Place 1 mL of urine in a tube.
Add two drops concentrated NH4OH.
Add 0.5 mL 5% silver nitrate.
Wait 10 minutes.
Add five drops sodium nitroprusside.
Observe for red-purple color.
Copyright © 2014. F.A. Davis Company
Heme Synthesis
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Porphyrin Disorders
• Intermediate compounds in the production of heme
• Primary porphyrins: uroporphyrin, coproporphyrin,
protoporphyrin
• Precursors: α-aminolevulinic acid (ALA) and porphobilinogen
• Detection of pathway disruptions in urine, blood, bile, and
feces
• Urine: ALA, porphobilinogen, urobilinogen
• Feces/bile: coproporphyrin, protoporphyrin
• Blood: free erythrocyte protoporphyrin (FEP) for lead
poisoning
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Porphyrin Disorders (cont’d)
• Collectively termed porphyrias
• Inherited: gene in metabolic pathway is missing
• Classified by clinical symptoms as
neurologic/psychiatric,
cutaneous/photosensitivity, or both
• Acquired (more common): lead poisoning,
alcoholism, iron deficiency, chronic liver and
renal disease
Copyright © 2014. F.A. Davis Company
Porphyrin Disorders (cont’d)
• Urine: port wine color after air exposure, also seen on
diapers
• Ehrlich reaction: only for ALA and porphobilinogen
– Convert ALA to porphobilinogen by adding acetyl acetone
– Watson-Schwartz test
– Ehrlich reaction now included on the Multistix urobilinogen
pad
• Fluorescence under ultraviolet light 550- to 600-nm range
is used for other porphyrins; extract into glacial acetic acid
and ethyl acetate; does not distinguish
– Violet, pink, red, based on concentration
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Mucopolysaccharide Disorders
• Inherited disorders preventing the metabolism of
glycoaminoglycans in the connective tissue
• Incompletely metabolized polysaccharides
accumulate in connective tissue
• Substances found in urine are dermatan sulfate,
keratan sulfate, and heparin sulfate
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Mucopolysaccharide Disorders
(cont’d)
• Mucopolysaccharidoses
– Hurler syndrome: abnormal skeletal structure, severe
mental retardation, and corneal damage
– Hunter syndrome: abnormal skeletal structure,
severe mental retardation, inherited as a sex-linked
recessive trait, rare in females
– Sanfilippo syndrome: mental retardation
• Bone marrow transplantation and gene therapy
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Urinary Screening Tests
• Acid-albumin and cetyltrimethylammonium
bromide turbidity tests
– Thick, white precipitate
• Metachromatic staining procedures
– Positive urine produces a blue color that cannot be
washed away with dilute acidified methanol
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Cetytrimethylammonium
Bromide (CTAB) Test
1. Place 5 mL of urine in a tube.
2. Add 1 mL 5% CTAB in citrate buffer.
3. Read turbidity in 5 minutes.
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Mucopolysaccharide Paper Test
1. Dip filter paper into 0.59% azure A dye in 2%
acetic acid.
2. Dry.
3. Add one drop of urine to paper.
4. Wash with 1 mL acetic acid + 200 mL methanol
diluted to a liter.
5. Observe for blue color.
Copyright © 2014. F.A. Davis Company
Purine Disorders
• Lesch-Nyhan disease
– Inherited sex-linked recessive
– Massive excretion of uric acid crystals
– Motor defects, mental retardation, self-destruction,
gout, renal calculi
– Normal development 6 to 8 months
– Orange sand in diaper
– Be alert for increased uric acid crystals in pediatric
patients
Copyright © 2014. F.A. Davis Company
Carbohydrate Disorders
• Termed melituria; frequently due to inherited disorder
• No problems except for galactosuria
• Three enzymes: most important is galactose-1-phosphate
uridyl transferase (GALT) also included in MS/MS screens
• Failure to thrive, severe mental retardation, cataracts, liver
disorders
• Remove lactose from diet
• Included in newborn testing of RBCs
• Clinitest positive
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Other Meliturias
• Lactosuria
– Pregnancy and lactation
• Fructosuria
– Parenteral feeding
– Resorcinol screening test
• Pentosuria
– Ingestion of large amounts of fruit
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