1. URINE SCREENING FOR METABOLIC DISORDERS
CHAPTER 8

2. Learning Objectives
Upon completing this chapter, the reader will be able to
Explain abnormal accumulation of metabolites in the urine in terms of overflow and renal disorders.
Discuss the importance of and the MS/MS testing methods for newborn screening.
Name the metabolic defect in phenylketonuria, and describe the clinical manifestations it produces.
State three causes of tyrosyluria.
Name the abnormal urinary substance present in alkaptonuria, and explain how its presence may be suspected.
Discuss the appearance and significance of urine that contains melanin.

3. Learning Objectives (cont’d)
Describe a basic laboratory observation that has relevance in maple syrup urine disease.
Discuss the significance of ketonuria in a newborn.
Differentiate between the presence of urinary indican owing to intestinal disorders and Hartnup disease.
State the significance of increased urinary 5-hydroxyindoleacetic acid.
Differentiate between cystinuria and cystinosis, including the differences found during analysis of the urine and the disease processes.

4. Learning Objectives (cont’d)
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.
Define mucopolysaccharides, and name three syndromes in which they are involved.
State the significance of increased uric acid crystals in newborns’ urine.
Explain the reason for performing tests for urinary-reducing substances on all newborns.

5. Overflow Versus Renal Disorders
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

6. Disorders Classified by Defect
Overflow Inherited
Metabolic
Renal
Phenylketonuria
Infantile tyrosinemia
Hartnup disease
Tyrosinemia
Melanuria
Cystinuria
Alkaptonuria
Indicanuria
Maple syrup urine disease
5-Hydroxyindoleacetic acid
Organic acidemias
Porphyria
Cystinosis
Mucopolysaccharidoses
Galactosemia
Lesch-Nyhan disease
Table 8–2 Major Disorders of Protein and Carbohydrate Metabolism Associated with Abnormal Urinary Constituents, Classified by Functional Defect

7. 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

8. Phenylalanine/Tyrosine Metabolic Pathway

9. 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)

10. Amino Acid Disorders (Aminoacidurias)(cont'd)
Phenylalanine hydroxylase is missing
Urine test
Urine and 5% ferric chloride produces a permanent green-blue color

11. 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

12. 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 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

13. Melanuria Second pathway for tyrosine Melanin
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

14. 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

15. Branched Chain Amino Acid Disorders
Amino acids with a methyl group that branches from the main aliphatic carbon chain
Two groups
Maple syrup urine disease (MSUD); early degradation products accumulate
Organic acidemias; accumulation of organic acids further down in pathway
Ketonuria in a newborn

16. 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

17. 2,4-Dinitrophenylhydrazine (DNPH) Test
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.

18. 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

19. Tryptophan Disorders
Increased urinary excretion of the metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA)

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. Cyanide-Nitroprusside Test
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.

27. 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

28. 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

29. 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

30. Silver Nitroprusside Test
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.

31. Heme Synthesis

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. Cetytrimethylammonium Bromide (CTAB) Test
Place 5 mL of urine in a tube.
Add 1 mL 5% CTAB in citrate buffer.
Read turbidity in 5 minutes.

39. Mucopolysaccharide Paper Test
Dip filter paper into 0.59% azure A dye in 2% acetic acid.
Dry.
Add one drop of urine to paper.
Wash with 1 mL acetic acid mL methanol diluted to a liter.
Observe for blue color.

40. 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

41. 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

42. Other Meliturias Lactosuria Fructosuria Pentosuria
Pregnancy and lactation
Fructosuria
Parenteral feeding
Resorcinol screening test
Pentosuria
Ingestion of large amounts of fruit