Copyright © 2014. F.A. Davis Company CHAPTER 8 URINE SCREENING FOR METABOLIC DISORDERS

Copyright © 2014. F.A. Davis Company

CHAPTER 8CHAPTER 8

URINE SCREENING FOR URINE SCREENING FOR METABOLIC DISORDERSMETABOLIC DISORDERS


Upon completing this chapter, the reader will be able to1.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.

Learning Objectives Learning Objectives


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 5-

hydroxyindoleacetic acid.11. Differentiate between cystinuria and cystinosis, including the

differences found during analysis of the urine and the disease processes.

Learning Objectives Learning Objectives (cont’d)(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.

Learning Objectives Learning Objectives (cont’d)(cont’d)


• 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

Overflow Versus Renal DisordersOverflow Versus Renal Disorders


Overflow Inherited Metabolic Renal

Phenylketonuria Infantile tyrosinemia Hartnup diseaseTyrosinemia Melanuria CystinuriaAlkaptonuria Indicanuria

Maple syrup urine disease 5-Hydroxyindoleacetic acid

Organic acidemias Porphyria

Cystinosis Porphyria Mucopolysaccharidoses

Galactosemia

Lesch-Nyhan disease

Disorders Classified by DefectDisorders Classified by Defect


• 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

Phenylalanine/Tyrosine Phenylalanine/Tyrosine Metabolic PathwayMetabolic Pathway


Phenylalanine/Tyrosine Phenylalanine/Tyrosine Metabolic PathwayMetabolic Pathway


• 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)

Amino Acid Disorders Amino Acid Disorders (Aminoacidurias) (Aminoacidurias)


• Phenylalanine hydroxylase is missing• Urine test

– Urine and 5% ferric chloride produces a permanent green-blue color

Amino Acid Disorders Amino Acid Disorders (Aminoacidurias)(cont'd)(Aminoacidurias)(cont'd)


• 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

Tyrosyluria/TyrosinemiaTyrosyluria/Tyrosinemia


• Hereditary defects– Type 2: enzyme deficiency is tyrosine

aminotransferase; corneal erosion and lesions on hands and feet

– Type 3: enzyme deficiency is p-hydroxyphenylpyruvate oxidase; mental retardation if no dietary restrictions (milk)

• Screening tests – Screening tests using MS/MS are available for

tyrosinemia types 1, 2, and 3

Tyrosyluria/Tyrosinemia Tyrosyluria/Tyrosinemia (cont’d)(cont’d)


• 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

MelanuriaMelanuria


• 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

AlkaptonuriaAlkaptonuria


• Amino acids with a methyl group that branches from the main aliphatic carbon chain

• Two groups1. Maple syrup urine disease (MSUD); early

degradation products accumulate2. Organic acidemias; accumulation of organic acids

further down in pathway• Ketonuria in a newborn

Branched Chain Branched Chain Amino Acid DisordersAmino Acid Disorders


• 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

Maple Syrup Urine Disease Maple Syrup Urine Disease (MSUD)(MSUD)


1. Place 1 mL 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 or white precipitate.

2,4-Dinitrophenylhydrazine 2,4-Dinitrophenylhydrazine (DNPH) Test(DNPH) Test


• 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

Organic AcidemiasOrganic Acidemias


Tryptophan DisordersTryptophan Disorders

• Increased urinary excretion of the metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA)


• 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

IndicanuriaIndicanuria


• Hartnup disease: blue diaper syndrome– Inherited disorder affects intestinal reabsorption of

indole and renal tubular reabsorption = Fanconi syndrome

– Requires dietary supplements: niacin

Indicanuria Indicanuria (cont’d)(cont’d)


• 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

5-Hydroxyindoleacetic 5-Hydroxyindoleacetic Acid (5-HIAA)Acid (5-HIAA)


• 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

5-Hydroxyindoleacetic 5-Hydroxyindoleacetic Acid (5-HIAA)(cont'd)Acid (5-HIAA)(cont'd)


• 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

Cystine DisordersCystine Disorders


• 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

Cystine Disorders Cystine Disorders (cont’d)(cont’d)


1. Place 3 mL of urine in a tube.2. Add 2 mL sodium cyanide.3. Wait 10 minutes.4. Add five drops 5% sodium nitroprusside.5. Observe for red-purple color.

Cyanide-Nitroprusside TestCyanide-Nitroprusside Test


• 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

CystinosisCystinosis


• 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

Cystinosis Cystinosis (cont’d)(cont’d)


• 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

HomocystinuriaHomocystinuria


1. Place 1 mL of urine in a tube.2. Add two drops concentrated NH4OH.3. Add 0.5 mL 5% silver nitrate.4. Wait 10 minutes.5. Add five drops sodium nitroprusside.6. Observe for red-purple color.

Silver Nitroprusside Test Silver Nitroprusside Test


Heme SynthesisHeme Synthesis


• 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

Porphyrin DisordersPorphyrin Disorders


• 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

Porphyrin Disorders Porphyrin Disorders (cont’d)(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

Porphyrin Disorders Porphyrin Disorders (cont’d)(cont’d)


• 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

Mucopolysaccharide DisordersMucopolysaccharide Disorders


• 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

Mucopolysaccharide Disorders Mucopolysaccharide Disorders (cont’d)(cont’d)


• 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

Urinary Screening TestsUrinary Screening Tests


1. Place 5 mL of urine in a tube.2. Add 1 mL 5% CTAB in citrate buffer.3. Read turbidity in 5 minutes.

Cetytrimethylammonium Cetytrimethylammonium Bromide (CTAB) TestBromide (CTAB) 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.

Mucopolysaccharide Paper TestMucopolysaccharide Paper Test


• 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

Purine DisordersPurine 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

Carbohydrate DisordersCarbohydrate Disorders


• Lactosuria– Pregnancy and lactation

• Fructosuria– Parenteral feeding– Resorcinol screening test

• Pentosuria – Ingestion of large amounts of fruit

Other MelituriasOther Meliturias

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Copyright © 2014. F.A. Davis Company CHAPTER 8 URINE SCREENING FOR METABOLIC DISORDERS