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CHAPTER 8
Urine Screening for
Metabolic Disorders
LEARNING OBJECTIVES
Upon completing this chapter, the reader will be able to:
8-1 Explain abnormal accumulation of metabolites in the 8-10 State the significance of increased urinary
urine in terms of overflow and renal disorders. 5-hydroxyindoleacetic acid.
8-2 Discuss the importance of and the MS/MS testing 8-11 Differentiate between cystinuria and cystinosis, in-
methods for newborn screening. cluding the differences found during analysis of the
urine and the disease processes.
8-3 Name the metabolic defect in phenylketonuria, and
describe the clinical manifestations it produces. 8-12 Describe the components in the heme synthesis path-
way, including the primary specimens used for their
8-4 State three causes of tyrosyluria.
analysis, and explain the cause and clinical signifi-
8-5 Name the abnormal urinary substance present in cance of major porphyrias and the appearance of
alkaptonuria, and explain how its presence may be porphyrins in urine.
suspected.
8-13 Define mucopolysaccharides, and name three syn-
8-6 Discuss the appearance and significance of urine that dromes in which they are involved.
contains melanin.
8-14 State the significance of increased uric acid crystals
8-7 Describe a basic laboratory observation that has rele- in newborns’ urine.
vance in maple syrup urine disease.
8-15 Explain the reason for performing tests for urinary-
8-8 Discuss the significance of ketonuria in a newborn. reducing substances on all newborns.
8-9 Differentiate between the presence of urinary indican
owing to intestinal disorders and Hartnup disease.

KEY TERMS
Alkaptonuria Inborn error of metabolism (IEM) Mucopolysaccharidoses
Cystinosis Indicanuria Organic acidemias
Cystinuria Lesch-Nyhan disease Phenylketonuria (PKU)
Galactosuria Maple syrup urine disease (MSUD) Porphyrinuria
Hartnup disease Melanuria Tyrosyluria
Homocystinuria Melituria
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164 Part Two | Urinalysis

As discussed in previous chapters, many of the abnormal re- renal type. Overflow disorders result from disruption of a nor-
sults obtained in the routine urinalysis are related to metabolic mal metabolic pathway that causes increased plasma concen-
rather than renal disease. Urine as an end product of body me- trations of the nonmetabolized substances. These chemicals
tabolism may contain additional abnormal substances not either override the reabsorption ability of the renal tubules or
tested for by routine urinalysis. Often these substances can be are not normally reabsorbed from the filtrate because they are
detected or monitored by additional screening tests that can present in only minute amounts. Abnormal accumulations of
also be performed in the urinalysis laboratory. Positive screen- the renal type are caused by malfunctions in the tubular reab-
ing tests can then be followed up with more sophisticated pro- sorption mechanism, as discussed in Chapter 7.
cedures performed in other sections of the laboratory. The most frequently encountered abnormalities are associ-
The need to perform additional tests may be detected by ated with metabolic disturbances that produce urinary overflow
the observations of alert laboratory personnel when performing of substances involved in protein, fat, and carbohydrate metab-
the routine analysis or from observations of abnormal speci- olism. This is understandable when one considers the vast num-
men color and odor by nursing staff and patients (Table 8–1). ber of enzymes used in the metabolic pathways of proteins, fats,
In other instances, clinical symptoms and family histories are and carbohydrates and the fact that their function is essential for
the deciding factors. complete metabolism. Disruption of enzyme function can be
caused by failure to inherit the gene to produce a particular
Overflow Versus Renal enzyme, referred to as an inborn error of metabolism (IEM),1
or by organ malfunction from disease or toxic reactions. The
Disorders most frequently encountered abnormal urinary metabolites are
summarized in Table 8–2, and their appearance is classified ac-
The appearance of abnormal metabolic substances in the urine cording to functional defect. Table 8–2 also includes substances
can be caused by a variety of disorders that can generally be and conditions that are covered in this chapter.
grouped into two categories, termed the overflow type and the

Newborn Screening Tests

Table 8–1 Abnormal Metabolic Constituents or
Conditions Detected in the Routine Many of the urine tests discussed in this chapter traditionally
Urinalysis were performed primarily to detect and monitor newborns for
IEMs. In recent years the screening of newborns has increased
Color Odor Crystals
to include more sensitive detection methods and ever-increasing
Homogentisic acid Phenylketonuria Cystine levels of state-mandated tests for IEMs. Many states currently
Melanin Maple syrup urine Leucine require testing for as many as 30 or more metabolic disorders.2
disease As discussed later in this chapter, because many of these
disorders cause the buildup of unmetabolized toxic food
Indican Isovaleric acidemia Tyrosine
ingredients, it is important that the defects be detected early
Porphyrins Cystinuria Lesch- in life. Levels of these substances are elevated more rapidly in
Cystinosis Nyhan blood than urine. Therefore, blood collected by infant heel
disease puncture is initially tested. Testing for many substances is
Homocystinuria
now performed using tandem mass spectrophotometry

Table 8–2 Major Disorders of Protein and Carbohydrate Metabolism Associated

With Abnormal Urinary Constituents, Classified by Functional 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
Porphyria
Mucopolysaccharidoses
Galactosemia
Lesch-Nyhan disease
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Chapter 8 | Urine Screening for Metabolic Disorders 165

(MS/MS). MS/MS is capable of screening the infant blood

sample for specific substances associated with particular
Amino Acid Disorders
IEMs. Figure 8–1 shows the standard form collected for test- The amino acid disorders with urinary screening tests include
ing using MS/MS. Methods for specific gene testing are also phenylketonuria (PKU), tyrosyluria, alkaptonuria, melanuria,
rapidly being developed. maple syrup urine disease, organic acidemias, indicanuria,
cystinuria, and cystinosis.

Phenylalanine-Tyrosine Disorders
Major inherited disorders include PKU, tyrosyluria, and
alkaptonuria. Metabolic defects cause overproduction of
melanin. The relationship of these varied disorders is illus-
trated in Figure 8–2.

Phenylketonuria
The most well known of the aminoacidurias, PKU is estimated
to occur in 1 of every 10,000 to 20,000 births and, if unde-
tected, results in severe mental retardation. It was first identified
in Norway by Ivan Følling in 1934, when a mother with other
Figure 8–1 Specimen collection form for MS/MS newborn screening mentally retarded children reported a peculiar mousy odor to
test. her child’s urine. Analysis of the urine showed increased

Normal Metabolism

Metabolic Disorders Phenylalanine Enzymes

Phenylketonuria Phenylalanine hydroxylase

Tyrosine
Phenylpyruvic acid
Tyrosine Melanin
aminotransferase Thyroxine Normal Byproducts
Tyrosinemia Epinephrine
Type 2
p-Hydroxyphenylpyruvic
acid
Tyrosyluria
p-Hydroxyphenylpyruvic acid p-Hydroxyphenylpyruvate
p-Hydroxyphenyllactic acid oxidase

Tyrosinemia Homogentisic acid

Type 3

Homogentisic acid
Alkaptonuria oxidase

Maleylacetoacetic
acid
Homogentisic
Maleylacetoacetic
acid
acid isomerase

Tyrosinemia Type 1b Fumarylacetoacetic

acid

Tyrosinemia Fumarylacetoacetic
Type 1 acid hydrolase

Tyrosinemia Type 1a Fumaric acid and

acetoacetic acid

Figure 8–2 Phenylalanine and tyrosine metabolic pathway including the normal pathway (blue), enzymes (yellow), and disorders caused by
failure to inherit particular enzymes (green).
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166 Part Two | Urinalysis

amounts of the keto acids, including phenylpyruvate. As shown of tyrosine metabolism may result from either inherited or meta-
in Figure 8–2, this occurs when the normal conversion of bolic defects. Also, because two reactions are directly involved
phenylalanine to tyrosine is disrupted. Interruption of the path- in the metabolism of tyrosine, the urine may contain excess ty-
way also produces children with fair complexions—even in rosine or its degradation products, p-hydroxyphenylpyruvic
dark-skinned families—owing to the decreased production of acid and p-hydroxyphenyllactic acid.
tyrosine and its pigmentation metabolite melanin. Most frequently seen is a transitory tyrosinemia in prema-
PKU is caused by failure to inherit the gene to produce the ture infants, which is caused by underdevelopment of the liver
enzyme phenylalanine hydroxylase. The gene is inherited as an function required to produce the enzymes necessary to com-
autosomal recessive trait with no noticeable characteristics or plete the tyrosine metabolism.
defects exhibited by heterozygous carriers. Fortunately, screen- Acquired severe liver disease also produces tyrosyluria re-
ing tests are available for early detection of the abnormality, and sembling that of the transitory newborn variety and, of course,
all states have laws that require the screening of newborns for is a more serious condition. In both instances, rarely seen ty-
PKU.2 Once discovered, dietary changes that eliminate phenyl- rosine and leucine crystals may be observed during micro-
alanine, a major constituent of milk, from the infant’s diet can scopic examination of the urine sediment.
prevent excessive buildup of serum phenylalanine, thereby Hereditary disorders in which enzymes required in the
avoiding damage to the child’s mental capabilities. As the child metabolic pathway are not produced present serious and often
matures, alternative pathways of phenylalanine metabolism de- fatal conditions that result in both liver and renal tubular dis-
velop, and dietary restrictions can be eased. Many products that ease producing a generalized aminoaciduria. Based on the en-
contain large amounts of phenylalanine, such as aspartame, zymes affected, the hereditary disorders can be classified into
now features warning labels for people with PKU. three types, all producing tyrosylemia and tyrosyluria.
The initial screening for PKU does not come under the aus- As shown in Figure 8–2, type 1 is caused by the deficiency
pices of the urinalysis laboratory, because increased blood levels of the enzyme fumarylacetoacetate hydrolase (FAH). Type 1
of phenylalanine must, of course, occur before urinary excretion produces a generalized renal tubular disorder and progressive
of phenylpyruvic acid, which may take 2 to 6 weeks. State laws liver failure in infants soon after birth. Type 2 tyrosinemia is
require that blood be collected after 24 hours after birth and be- caused by lack of the enzyme tyrosine aminotransferase. Per-
fore the newborn leaves the hospital. The increasing tendency to sons develop corneal erosion and lesions on the palms, fingers,
release newborns from the hospital as early as 24 hours after birth and soles of the feet believed to be caused by crystallization of
has caused concern about the ability to detect increased phenyl- tyrosine in the cells. Type 3 tyrosinemia is caused by lack of
alanine levels at that early stage. Studies have shown that in many the enzyme p-hydroxyphenylpyruvic acid dioxygenase. This
cases phenylalanine can be detected as early as 4 hours after birth can result in mental retardation if dietary restrictions of pheny-
and, if the cutoff level for normal results is lowered from 4 mg/dL lalanine and tyrosine are not implemented.
to 2 mg/dL, the presence of PKU should be detected. Tests may Screening tests using MS/MS are available for tyrosinemia
need to be repeated during an early visit to the pediatrician. More types 1, 2, and 3. See Procedure 8-2 for urine testing for tyro-
girls than boys escape detection of PKU during early tests because syluria using nitroso-naphthol.
of slower rises in blood phenylalanine levels.1
Melanuria
Urine testing using ferric chloride may be used as a follow-
up test to ensure proper dietary control in previously diagnosed The previous discussion focused on the major phenylalanine-
cases and as a means of monitoring the dietary intake of preg- tyrosine metabolic pathway illustrated in Figure 8–2; how-
nant women known to lack phenylalanine hydroxylase. ever, as also shown in Figure 8–2 and is the case with many
Urine tests for phenylpyruvic acid are based on the ferric amino acids, a second metabolic pathway also exists for
chloride reaction performed by tube test. The addition of ferric tyrosine. This pathway is responsible for the production of
chloride to urine containing phenylpyruvic acid produces a
permanent blue-green color (see Procedure 8-1).

Tyrosyluria PROCEDURE 8-2

The accumulation of excess tyrosine in the plasma (tyrosinemia) Nitroso-Naphthol Test for Tyrosine
producing urinary overflow may be due to several causes and
is not well categorized. As can be seen in Table 8–2, disorders 1. Place five drops of urine in a tube.
2. Add 1 mL of 2.63N nitric acid.
3. Add one drop of 21.5% sodium nitrite.
PROCEDURE 8-1
4. Add 0.1 mL 1-nitroso-2-napthol.
Ferric Chloride Tube Test 5. Mix.
1. Place 1 mL of urine in a tube. 6. Wait 5 minutes.
2. Slowly add five drops of 10% ferric chloride. 7. Observe for an orange-red color, indicating tyrosine
3. Observe color for a permanent blue-green color. metabolites.
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Chapter 8 | Urine Screening for Metabolic Disorders 167

melanin, thyroxine, epinephrine, protein, and tyrosine sul-

PROCEDURE 8-3
fate. Of these substances, the major concern of the urinalysis
laboratory is melanin, the pigment responsible for the dark Homogentisic Acid Test
color of hair, skin, and eyes. Deficient production of melanin
1. Place 4 mL of 3% silver nitrate in a tube.
results in albinism.
Increased urinary melanin darkens the urine. The dark- 2. Add 0.5 mL of urine.
ening appears after the urine is exposed to air. Elevated urinary 3. Mix.
melanin is a serious finding that indicates proliferation of the 4. Add 10% NH4OH by drops.
normal melanin-producing cells (melanocytes), producing a
5. Observe for black color.
malignant melanoma. These tumors secrete a colorless precur-
sor of melanin, 5,6-dihydroxyindole, which oxidizes to
melanogen and then to melanin, producing the characteristic
dark urine.

Alkaptonuria TECHNICAL TIP It is important to differentiate between

the presence of homogentisic acid and melanin when
Alkaptonuria was one of the six original inborn errors of me- urine is observed to have turned black upon standing.
tabolism described by Garrod in 1902. The name alkaptonuria
was derived from the observation that urine from patients with
this condition darkened after becoming alkaline from standing
at room temperature. Therefore, the term “alkali lover,” or Branched-Chain Amino Acid Disorders
alkaptonuria, was adopted. This metabolic defect is actually
the third major defect in the phenylalanine-tyrosine pathway The branched-chain amino acids differ from other amino acids
and occurs from failure to inherit the gene to produce the by having a methyl group that branches from the main
enzyme homogentisic acid oxidase. Without this enzyme, the aliphatic carbon chain (Fig. 8–3 A and B). Two major groups
phenylalanine-tyrosine pathway cannot proceed to comple- of disorders are associated with errors in the metabolism of the
tion, and homogentisic acid accumulates in the blood, tissues, branched-chain amino acids. In one group, accumulation of
and urine. This condition does not usually manifest itself clin- one or more of the early amino acid degradation products oc-
ically in early childhood, but observations of brown-stained curs, as is seen in maple syrup urine disease. Disorders in the
or black-stained cloth diapers and reddish-stained disposable other group are termed organic acidemias and result in accu-
diapers have been reported.3 In later life, brown pigment be- mulation of organic acids produced further down in the amino
comes deposited in the body tissues (particularly noticeable acid metabolic pathway.
in the ears). Deposits in the cartilage eventually lead to arthri- A significant laboratory finding in branched-chain amino
tis. A high percentage of persons with alkaptonuria develop acid disorders is the presence of ketonuria in a newborn.
liver and cardiac disorders.4 Maple Syrup Urine Disease
Homogentisic acid reacts in several of the routinely used
screening tests for metabolic disorders, including the ferric Although maple syrup urine disease (MSUD) is rare, a brief
chloride test, in which a transient deep blue color is produced discussion is included in this chapter because the urinalysis
in the tube test. A yellow precipitate is produced in the Clin- laboratory can provide valuable information for the essential
itest, indicating the presence of a reducing substance. Another early detection of this disease. MSUD is also included in new-
screening test for urinary homogentisic acid is to add alkali to born screening profiles using MS/MS.
freshly voided urine and to observe for darkening of the color; MSUD is caused by an IEM, inherited as an autosomal
however, large amounts of ascorbic acid interfere with this recessive trait. The amino acids involved are leucine,
reaction. isoleucine, and valine. The metabolic pathway begins nor-
Paper and thin layer chromatography procedures are avail- mally, with the transamination of the three amino acids in the
able for quantitating homogentisic acid. The silver nitrate test liver to the keto acids (α -ketoisovaleric, α -ketoisocaproic,
for homogentisic acid is provided in Procedure 8-3. and α -keto-β -methylvaleric). Failure to inherit the gene for
the enzyme necessary to produce oxidative decarboxylation
of these keto acids results in their accumulation in the blood
and urine.1
TECHNICAL TIP The appearance of black urine from a
Newborns with MSUD begin to exhibit clinical symptoms
patient of any age should be reported to a supervisor.
associated with failure to thrive after approximately l week. The
presence of the disease may be suspected from these clinical
symptoms; however, many other conditions have similar symp-
toms. Personnel in the urinalysis laboratory or in the nursery
TECHNICAL TIP Melanin may react with sodium nitro- may detect the disease by noticing a urine specimen that pro-
ferricyanide (acetone reagent strip), producing a red color. duces a strong odor resembling maple syrup that is caused by
the rapid accumulation of keto acids in the urine. Even though
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168 Part Two | Urinalysis

Alpha Amino Group Alpha Amino Group Carboxyl Group

H H O H H O

N C C N C C
Figure 8–3 α -Alpha amino acid
and branched chain amino acid
H R H CH structures. A. Structure of an
OH OH
α -amino acid. B. Structure of
R Group H 3C CH3 the branched chain amino acid
(Variant)
A B Leucine Group leucine.

a report of urine odor is not a part of the routine urinalysis, no- Figure 8–4 shows a simplified diagram of the metabolic path-
tifying the physician about this unusual finding can prevent ways by which these substances are produced. Other metabolic
progression to severe mental retardation and even death. Stud- pathways of tryptophan are not included because they do not
ies have shown that if maple syrup urine disease is detected by relate directly to the urinalysis laboratory.
the 11th day, dietary regulation and careful monitoring of
urinary keto acid concentrations can control the disorder.5 Indicanuria
The 2,4-dinitrophenylhydrazine (DNPH) urine screening test Under normal conditions, most of the tryptophan that enters
for MSUD is provided in Procedure 8-4. the intestine is either reabsorbed for use by the body in pro-
ducing protein or is converted to indole by intestinal bacteria
Organic Acidemias and excreted in the feces. However, in certain intestinal dis-
Generalized symptoms of the organic acidemias include early orders (including obstruction; the presence of abnormal bac-
severe illness, often with vomiting accompanied by metabolic teria; malabsorption syndromes; and Hartnup disease, a rare
acidosis; hypoglycemia; ketonuria; and increased serum am- inherited disorder, increased amounts of tryptophan are con-
monia.6 The three most frequently encountered disorders are verted to indole. The excess indole is then reabsorbed from
isovaleric, propionic, and methylmalonic acidemia. the intestine into the bloodstream and circulated to the liver,
Isovaleric acidemia may be suspected when urine speci- where it is converted to indican and then excreted in the
mens, and sometimes even the patient, possess a characteristic urine. Indican excreted in the urine is colorless until oxidized
odor of “sweaty feet.” This odor is caused by the accumulation to the dye indigo blue by exposure to air. Early diagnosis of
of isovalerylglycine due to a deficiency of isovaleryl coenzyme Hartnup disease is sometimes made when a mother reports a
A in the leucine pathway. blue staining of her infant’s diapers, referred to as the “blue
The presence of isovaleric, propionic, and methylmalonic diaper syndrome.”
acidemias can be detected by newborn screening programs Except in cases of Hartnup disease, correction of the un-
using MS/MS. derlying intestinal disorder returns urinary indican levels to
Propionic and methylmalonic acidemias result from errors normal. The inherited defect in Hartnup disease affects not
in the metabolic pathway converting isoleucine, valine, threo- only the intestinal reabsorption of tryptophan but also the
nine, and methionine to succinyl coenzyme A. Propionic renal tubular reabsorption of other amino acids, resulting in
acid is the immediate precursor to methylmalonic acid in this a generalized aminoaciduria (Fanconi syndrome). The defec-
pathway. tive renal transport of amino acids does not appear to affect
other renal tubular functions. Therefore, with proper dietary
Tryptophan Disorders supplements, including niacin, people with Hartnup disease
The major concern of the urinalysis laboratory in the metabo- have a good prognosis.7
lism of tryptophan is the increased urinary excretion of the 5-Hydroxyindoleacetic Acid
metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA).
As shown in Figure 8–4, a second metabolic pathway of
tryptophan is for the production of serotonin used in the
PROCEDURE 8-4 stimulation of smooth muscles. Serotonin is produced from
tryptophan by the argentaffin cells in the intestine and is car-
2,4-Dinitrophenylhydrazine (DNPH) Test for MSUD ried through the body primarily by the platelets. Normally,
the body uses most of the serotonin, and only small amounts
1. Place 1 mL of urine in a tube.
of its degradation product, 5-HIAA, are available for excre-
2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl. tion in the urine. However, when carcinoid tumors involving
3. Wait 10 minutes. the argentaffin (enterochromaffin) cells develop, excess
4. Observe for yellow turbidity or precipitate. amounts of serotonin are produced, resulting in the elevation
of urinary 5-HIAA levels.
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Chapter 8 | Urine Screening for Metabolic Disorders 169

Tryptophan
(normal)
Abnormal Abnormal
Intestinal disorder Malignant tumors
Excess indole 5-Hydroxytryptophan
Reabsorbed from intestine
into bloodstream
Liver

Indican Indole Serotonin

Urine

Exposure to air

Indigo blue Feces 5-Hydroxyindoleacetic

acid (5-HIAA)
Figure 8–4 Tryptophan metabolism.

Adding nitrous acid and 1-nitroso-2-naphthol to urine that of cystine. It is now known that although both disorders are
contains 5-HIAA causes the urine to turn purple to black, de- inherited, one is a defect in the renal tubular transport of amino
pending on the amount of 5-HIAA present (Procedure 8-5). The acids (cystinuria) and the other is an IEM (cystinosis). A no-
normal daily excretion of 5-HIAA is 2 to 8 mg, and excretion of ticeable odor of sulfur may be present in the urine of people
greater than 25 mg/24 h can be an indication of argentaffin cell with cystine metabolism disorders.
tumors.8 The test can be performed on a random or first morn-
ing specimen; however, false-negative results can occur based Cystinuria
on the specimen concentration and also because 5-HIAA As the name implies, cystinuria is marked by elevated amounts
may not be produced at a constant rate throughout the day. If a of the amino acid cystine in the urine. The presence of in-
24-hour sample is used, it must be preserved with hydrochloric creased urinary cystine is not due to a defect in the metabolism
or boric acid. A plasma method using high-performance liquid of cystine but, rather, to the inability of the renal tubules to re-
chromatography with fluorescence detection is also available. absorb cystine filtered by the glomerulus. The demonstration
Patients must be given explicit dietary instructions before that not only cystine but also lysine, arginine, and ornithine
collecting any sample to be tested for 5-HIAA, because serotonin are not reabsorbed has ruled out the possibility of an error
is a major constituent of foods such as bananas, pineapples, and in metabolism even though the condition is inherited.9 The
tomatoes. Medications, including phenothiazines and acetanilids, disorder has two modes of inheritance: one in which reabsorp-
also interfere with results. Patients should be directed to withhold tion of all four amino acids—cystine, lysine, arginine, and
medications for 72 hours before specimen collection. ornithine—is affected, and the other in which only cystine and
lysine are not reabsorbed. Genetic studies have grouped cystin-
Cystine Disorders uria into three types based on the two inherited genes and their
Two distinct disorders of cystine metabolism exhibit renal heterozygous and homozygous inheritance. In general, persons
manifestations. Confusion as to their relationship existed for with any form of inheritance may form renal calculi but the
many years following the discovery of renal calculi consisting calculi are less common in persons in whom only lysine and
cystine are affected.10 Approximately 65% of the people in
whom all four amino acids are affected can be expected to pro-
PROCEDURE 8-5 duce calculi early in life.
Because cystine is much less soluble than the other three
Silver Nitroprusside Test amino acids, laboratory screening determinations are based on
1. Place 1 mL of urine in a tube. observing cystine crystals in the sediment of concentrated or
2. Add two drops concentrated NH4OH. first morning specimens. Cystine is also the only amino acid
found during the analysis of calculi from these patients. A
3. Add 0.5 mL 5% silver nitrate.
chemical screening test for urinary cystine can be performed
4. Wait 10 minutes. using cyanide-nitroprusside. Reduction of cystine by sodium
5. Add five drops sodium nitroprusside. cyanide followed by the addition of nitroprusside produces
6. Observe for purple-black color. a red-purple color in a specimen that contains excess cystine
(see Procedure 8-6). False-positive reactions occur in the
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170 Part Two | Urinalysis

an additional screening test for homocystinuria must be per-

PROCEDURE 8-6
formed by following a positive cyanide-nitroprusside test
Cyanide-Nitroprusside Test for Cystine result with a silver-nitroprusside test, in which only homocys-
tine will react. The use of silver nitrate in place of sodium
1. Place 3 mL of urine in a tube.
cyanide reduces homocystine to its nitroprusside-reactive
2. Add 2 mL sodium cyanide. form but does not reduce cystine. Consequently, a positive re-
3. Wait 10 minutes. action in the silver-nitroprusside test confirms the presence of
4. Add five drops 5% sodium nitroprusside. homocystinuria. Fresh urine should be used when testing for
homocystine (see Procedure 8-7).
5. Observe for red-purple color.

Porphyrin Disorders
presence of ketones and homocystine, and additional tests may Porphyrins are the intermediate compounds in the production
have to be performed. of heme. The basic pathway for heme synthesis presented in
Figure 8–5 shows the three primary porphyrins (uropor-
Cystinosis phyrin, coproporphyrin, and protoporphyrin) and the por-
Regarded as a genuine IEM, cystinosis can occur in three vari- phyrin precursors (α -aminolevulinic acid [ALA] and
ations, ranging from a severe fatal disorder developed in infancy porphobilinogen). As can be seen, the synthesis of heme can
to a benign form appearing in adulthood. The disorder has two be blocked at a number of stages. Blockage of a pathway re-
general categories, termed nephropathic and nonnephropathic. action results in the accumulation of the product formed just
The nephropathic category is subdivided into infantile and late- before the interruption. Detection and identification of this
onset cystinosis. A defect in the lysosomal membranes prevents product in the urine, bile, feces, or blood can then aid in de-
the release of cystine into the cellular cytoplasm for metabolism. termining the cause of a specific disorder.
The incomplete metabolism of cystine results in crystalline The solubility of the porphyrin compounds varies with
deposits of cystine in many areas of the body, including the their structure. ALA, porphobilinogen, and uroporphyrin are
cornea, bone marrow, lymph nodes, and internal organs. the most soluble and readily appear in the urine. Copropor-
A major defect in the renal tubular reabsorption mechanism phyrin is less soluble but is found in the urine, whereas proto-
(Fanconi syndrome) also occurs. The renal tubules, particularly porphyrin is not seen in the urine. Fecal analysis has usually
the proximal convoluted tubules, are affected by the cystine de- been performed to detect coproporphyrin and protoporphyrin.
posits that interfere with reabsorption. This is not an inherited However, to avoid false-positive interference, bile is a more ac-
disorder of renal tubular reabsorption, as seen in cystinuria. ceptable specimen.11 The Centers for Disease Control and Pre-
Continued deposition of cystine, if untreated, results in renal vention (CDC) recommends analysis of whole blood for the
failure early in life. In infantile nephropathic cystinosis, there is presence of free erythrocyte protoporphyrin (FEP) as a screen-
rapid progression to renal failure. In late-onset nephropathic ing test for lead poisoning.
cystinosis, there is a gradual progression to total renal failure. Disorders of porphyrin metabolism are collectively termed
Renal transplants and the use of cystine-depleting medications porphyrias. They can be inherited or acquired from erythro-
to prevent the buildup of cystine in other tissues are extending cytic and hepatic malfunctions or exposure to toxic agents.
lives. Nonnephropathic cystinosis is relatively benign but may Common causes of acquired porphyrias include lead poison-
cause some ocular disorders. ing, excessive alcohol intake, iron deficiency, chronic liver dis-
Routine laboratory findings in infantile nephropathic ease, and renal disease. Inherited porphyrias are much rarer
cystinosis include polyuria, generalized aminoaciduria, positive than acquired porphyrias. They are caused by failure to inherit
Clinitest results for reducing substances, and lack of urinary the gene that produces an enzyme needed in the metabolic
concentration. pathway. The enzyme deficiency sites for some of the more

Homocystinuria
Defects in the metabolism of the amino acid methionine pro- PROCEDURE 8-7
duce an increase in homocystine throughout the body. The
increased homocystine can result in failure to thrive, cataracts, Silver Nitroprusside Test for Homocystine
mental retardation, thromboembolic problems, and death. 1. Place 1 mL of urine in a tube.
Early detection of this disorder (homocystinuria) and a change 2. Add two drops concentrated NH4OH.
in diet that excludes foods high in methionine can alleviate the
3. Add 0.5 mL 5% silver nitrate.
metabolic problems. Therefore, screening for homocystine is
included in newborn screening programs. Newborn screening 4. Wait 10 minutes.
tests are performed using MS/MS testing. 5. Add five drops sodium nitroprusside.
As mentioned, increased urinary homocystine gives a 6. Observe for red-purple color.
positive result with the cyanide-nitroprusside test. Therefore,
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Chapter 8 | Urine Screening for Metabolic Disorders 171

HEME SYNTHESIS

Glycine and Succinyl-CoA

Aminolevulinic acid synthetase

γ-Aminolevulinate (ALA)

Aminolevulinic acid synthetase Lead exposure

ALA hydratase deficiency porphyria

Porphobilinogen

Uroporphyrinogen synthase

Acute intermittent porphyria

Hydroxymethylbilane

Uroporphyrinogen cosynthase

Congenital erythropoietic porphyria

Uroporphyrinogen (UPG)

Uroporphyrinogen decarboxylase

Porphyria cutanea tarda

Coproporphyrinogen (CPG)

Coproporphyrinogen oxidase

Hereditary coproporphyria

Protoporphyrinogen

Protoporphyrinogen oxidase

Variate porphyria

Protoporphyrin IX

Ferrochelatase Lead exposure

Erythropoietic protoporphyria
Figure 8–5 Pathway of heme formation, including normal
pathway (green), enzymes (orange), and stages affected by Heme
the major disorders (yellow) of porphyrin metabolism.

common porphyrias are shown in Figure 8–5. The inherited detection of ALA and porphobilinogen. Acetyl acetone must
porphyrias are frequently classified by their clinical symptoms, be added to the specimen to convert the ALA to porphobilino-
either neurologic/psychiatric or cutaneous photosensitivity or gen prior to performing the Ehrlich test. The fluorescent tech-
a combination of both (Table 8–3). nique must be used for the other porphyrins. The Ehrlich
An indication of the possible presence of porphyrinuria reaction that is now included in the Multistix urobilinogen pad
is the observation of a red or port wine color to the urine after was originally used for all urobilinogen testing. Variations of
exposure to air. The port wine urine color is more prevalent in the Ehrlich reaction include the Watson-Schwartz test for dif-
the erythropoietic porphyrias, and staining of the teeth may ferentiation between the presence of urobilinogen and porpho-
also occur. As seen with other inherited disorders, the presence bilinogen (see Procedures 8-8 and 8-9) and the Hoesch test
of congenital porphyria is sometimes suspected from a red dis- (Procedure 8-10).
coloration of an infant’s diapers. Testing for the presence of porphobilinogen is most useful
The two screening tests for porphyrinuria use the Ehrlich when patients exhibit symptoms of an acute attack. This can
reaction and fluorescence under ultraviolet light in the 550- to be done with the Hoesch test. Increased porphobilinogen is
600-nm range. The Ehrlich reaction can be used only for the associated with acute intermittent porphyria. A negative test
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172 Part Two | Urinalysis

Table 8–3 Common Porphyrias

Porphyria Elevated Compound(s) Clinical Symptoms Laboratory Testing

Acute intermittent porphyria ALA Porphobilinogen Neurologic/psychiatric Urine/Ehrlich reaction

Porphyria cutanea tarda Uroporphyrin Photosensitivity Urine fluorescence
Congenital erythropoietic Uroporphyrin Photosensitivity Urine or feces fluorescence
porphyria Coproporphyrin
Variegate porphyria Coproporphyrin Photosensitivity/ Bile or feces fluorescence
neurologic
Erythropoietic protoporphyria Protoporphyrin Photosensitivity Blood FEP
Bile or feces fluorescence
Lead poisoning ALA Neurologic Acetoacetic acid + urine/
Ehrlich reaction
Protoporphyrin Blood FEP

HISTORICAL NOTE Mucopolysaccharide

Vampires in Old Europe Disorders
Mucopolysaccharides, or glycosaminoglycans, are a group
Did you ever wonder how the legend of vampires got of large compounds located primarily in the connective tis-
started? Think about the previous discussion on the symp- sue. They consist of a protein core with numerous polysac-
toms and inheritance of porphyrias. charide branches. Inherited disorders in the metabolism of
Photosensitivity→ Avoidance of sunlight these compounds prevent complete breakdown of the poly-
Pale coloring→ Anemia caused by heme disorder saccharide portion of the compounds, resulting in accumu-
Port wine− colored urine, red-stained teeth→ Drinking lation of the incompletely metabolized polysaccharide
blood portions in the lysosomes of the connective tissue cells and
Psychiatric symptoms→ Abnormal behavior their increased excretion in the urine. The products most
Inherited disorder→ Familial association, small gene frequently found in the urine are dermatan sulfate, keratan
pool sulfate, and heparan sulfate, with the appearance of a partic-
Dracula is associated with Transylvania, now Romania. ular substance being determined by the specific metabolic
Porphyria was a common disease of early royalty in error that was inherited.12 Therefore, identification of the
Europe as a result of intermarriage among the royals of specific enzyme deficiency is necessary to establish a specific
different countries. King George III reportedly died blind, diagnosis.
deaf, and mad from porphyria. There are many types of mucopolysaccharidoses, but
the best known are Hurler syndrome, Hunter syndrome, and
Sanfilippo syndrome. In both Hurler and Hunter syndromes,
result is obtained in the presence of lead poisoning unless ALA the skeletal structure is abnormal and there is severe mental
is first converted to porphobilinogen. retardation; in Hurler syndrome, mucopolysaccharides accu-
Fluorescent screening for the other porphyrins uses mulate in the cornea of the eye. Hunter syndrome is inherited
their extraction into a mixture of glacial acetic acid and ethyl as sex-linked recessive and is rarely seen in females. Without
acetate. The solvent layer is then examined. Negative reac- treatment, both syndromes are usually fatal during childhood,
tions have a faint blue fluorescence. Positive reactions fluo- whereas in Sanfilippo syndrome, the only abnormality is men-
resce as violet, pink, or red, depending on the concentration tal retardation. Bone marrow transplants and gene replace-
of porphyrins. If the presence of interfering substances is ment therapy are the most promising treatments for these
suspected, the organic layer can be removed to a separate disorders.
tube, and 0.5 mL of hydrochloric acid added to the tube. Urinary screening tests for mucopolysaccharides may
Only porphyrins are extracted into the acid layer, which then be requested either as part of a routine battery of tests
produces a bright orange-red fluorescence. The fluorescence performed on all newborns or on infants who exhibit symp-
method does not distinguish among uroporphyrin, copro- toms of mental retardation or failure to thrive. The most
porphyrin, and protoporphyrin, but it rules out porpho- frequently used screening tests are the acid-albumin and
bilinogen and ALA. Identifying specific porphyrins requires cetyltrimethylammonium bromide (CTAB) turbidity tests
additional techniques and the analysis of fecal and erythro- and the metachromatic staining spot tests. In both the
cyte samples. Increased protoporphyrin is best measured in acid-albumin and the CTAB tests, a thick, white turbidity
whole blood. forms when these reagents are added to urine that contains
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Chapter 8 | Urine Screening for Metabolic Disorders 173

PROCEDURE 8-8 PROCEDURE 8-9

Watson-Schwartz Differentiation Test Watson-Schwartz Test
The classic test for differentiating between urobilinogen, 1. Label two tubes #1 and #2.
porphobilinogen, and Ehrlich-reactive compounds is the 2. To each tube add:
Watson-Schwartz test. The test is performed as follows:
Tube 1 Tube 2
1. To each tube add:
2 mL urine 2 mL urine
Tube 1 Tube 2
2 mL chloroform 2 mL butanol
2 mL urine 2 mL urine
4 mL sodium acetate 4 mL sodium acetate
2 mL chloroform 2 mL butanol
3. Vigorously shake both tubes.
4 mL sodium acetate 4 mL sodium acetate
4. Place in a rack for layers to settle.
2. Observe the color of the layers.
5. Observe both tubes for red color in the layers.
3. Interpretation:
Interpretation:
The addition of chloroform to Tube 1 results in the ex-
Tube 1
traction of urobilinogen into the chloroform (bottom) layer,
producing a colorless urine (top) layer, and a red chloroform Upper layer = urine; if colorless = porphobilinogen or
layer on the bottom. Neither porphobilinogen nor other Ehrlich-reactive compounds.
Ehrlich-reactive compounds are soluble in chloroform. Bottom layer = chloroform; if red = urobilinogen.
Porphobilinogen is also not soluble in butanol; however, If both layers are red, re-extract the urine layer from
urobilinogen and other Ehrlich-reactive compounds are ex- tube 1.
tracted into butanol. Therefore, the addition of butanol to
Place 2 mL of urine layer from Tubes 1 and 2 mL chloro-
Tube 2 produces a red (upper) butanol layer if urobilinogen
form and 4 mL sodium acetate into a new tube. Repeat
or Ehrlich-reactive compounds are present and a colorless
procedure.
butanol layer if porphobilinogen is present. As shown in the
figure, urobilinogen is soluble in both chloroform and Interpretation: Upper layer—urine colorless
butanol, and porphobilinogen is soluble in neither. If both Bottom layer—chloroform—red = excess urobilinogen
urobilinogen and porphobilinogen are present, both layers Both layers red = porphobilinogen and urobilinogen
appear red. Before reporting the test as positive for both
Tube 2
substances, an additional chloroform extraction should be
performed on the red urine (upper) layer in Tube 1 to ensure Upper layer = butanol If red = urobilinogen or
that the red color is not due to excess urobilinogen. Ehrlich-reactive compounds
Bottom layer = urine If colorless = porphobilinogen

UR B UR B UR B
PROCEDURE 8-10

C UR C UR C UR
Hoesch Screening Test for Porphobilinogen
The Hoesch test is used for rapid screening or monitoring
of urinary porphobilinogen.
Urobilinogen Porphobilinogen Ehrlich-reactive
1. Two drops of urine are added to approximately 2 mL of
Hoesch reagent (Ehrlich reagent dissolved in 6 M HCl).
2. Immediately observed the top of the solution for the
appearance of a red color that indicates the presence
of porphobilinogen.
3. Shake the tube.
UR B UR UR
Interpretation:
When the tube is shaken, the red color is seen throughout
C UR C C the solution. The test detects approximately 2 mg/dL of
porphobilinogen, and urobilinogen is inhibited by the
Urobilinogen/porphobilinogen Excess urobilinogen
highly acidic pH. High concentrations of methyldopa and
indican, and highly pigmented urines, may produce false-
Watson-Schwartz reactions. positive results.
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174 Part Two | Urinalysis

mucopolysaccharides. Turbidity is usually graded on a scale Galactosuria can be caused by a deficiency in any of three
of 0 to 4 after 30 minutes with acid-albumin and after enzymes, galactose-1-phosphate uridyl transferase (GALT),
5 minutes with CTAB.13 (See Procedure 8-11.) galactokinase, and UDP-galactose-4-epimerase. Of these en-
zymes, it is GALT deficiency that causes the severe, possibly fatal
symptoms associated with galactosemia. Newborn screening
Purine Disorders protocols currently test for the presence of GALT deficiency. The
enzyme is measured in the red blood cells as part of the newborn
A disorder of purine metabolism known as Lesch-Nyhan
heel puncture protocol. As a result, people with deficiencies in
disease that is inherited as a sex-linked recessive results in
the other two enzymes may still produce galactosuria but have
massive excretion of urinary uric acid crystals. Failure to in-
negative newborn screening tests. Galactose kinase deficiency
herit the gene to produce the enzyme hypoxanthine guanine
can result in cataracts in adulthood. UDP-galactose-4-epimerase
phosphoribosyltransferase is responsible for the accumulation
deficiency may be asymptomatic or produce mild symptoms.
of uric acid throughout the body. Patients suffer from severe
Other causes of melituria include lactose, fructose, and
motor defects, mental retardation, a tendency toward self-
pentose. Lactosuria may be seen during pregnancy and lacta-
destruction, gout, and renal calculi. Development is usually
tion. Fructosuria is associated with parenteral feeding and
normal for the first 6 to 8 months, and the first sign is often
pentosuria with ingestion of large amounts of fruit. Additional
uric acid crystals resembling orange sand in diapers.7 Labora-
tests including chromatography can be used to identify other
tories should be alert for the presence of increased uric acid
nonglucose reducing substances.
crystals in pediatric urine specimens.

Carbohydrate Disorders Log on to

[Link]/strasinger
for additional content related
The presence of increased urinary sugar (melituria) is most to this chapter.
frequently due to an inherited disorder. In fact, pentosuria was
one of Garrod’s original six IEMs.14 Fortunately, most meli-
turias cause no disturbance to body metabolism. However, as References
discussed in Chapter 5, pediatric urine should be routinely 1. Frimpton, GW: Aminoacidurias due to inherited disorders of
screened for the presence of reducing substances using the metabolism. N Engl J Med 1289:835–901, 1973.
2. National newborn screening and genetics resource center. Web
Clinitest procedure. The finding of a positive copper reduction site: http:// [Link] Accessed January 17,
test result combined with a negative reagent strip glucose oxi- 2007.
dase test result is strongly suggestive of a disorder of carbohy- 3. Nyhan, WL, and Sakati, NO: Diagnostic Recognition of Genetic
drate metabolism. Of primary concern is the presence of Disease. Lea & Febiger, Philadelphia, 1987.
galactosuria, indicating the inability to properly metabolize 4. Stanbury, JB: The Metabolic Basis of Inherited Diseases.
McGraw-Hill, New York, 1983.
galactose to glucose. The resulting galactosemia with toxic in- 5. Clow, CL, Reade, TH, and Scriver, CR: Outcome of early and
termediate metabolic products results in infant failure to thrive, long-term management of classical maple syrup urine disease.
combined with liver disorders, cataracts, and severe mental re- Pediatrics 68(6):856–862, 1981.
tardation. Early detection of galactosuria followed by removal 6. Goodman, SI: Disorders of organic acid metabolism. In Emery,
of lactose (a disaccharide containing galactose and glucose) AEH, and Rimoin, DL: Principles and Practice of Medical
Genetics. Churchill Livingstone, New York, 1990.
from the diet can prevent these symptoms. 7. Jepson, JB: Hartnup’s disease. In Stanbury, JB, Wyngaarden, JB,
and Fredrickson, DS (eds): The Metabolic Basis of Inherited
Diseases. McGraw-Hill, New York, 1983.
8. Van Leeuwen, AM, Poelhuis-Leth, DJ, and Bladh, ML: Davis’s
PROCEDURE 8-11 Comprehensive Laboratory and Diagnostics Handbook. 4th ed.
Philadelphia, FA Davis Company; 2011
9. Nyhan, WL: Abnormalities in Amino Acid Metabolism in Clini-
Cetyltrimethylammonium Bromide (CTAB) Test for
cal Medicine. Appleton-Century-Crofts, Norwalk, CT., 1984.
Mucopolysaccharides 10. Dello Strolongo, L, et al: Comparison between SLC3A1 and
1. Place 5 mL of urine in a tube. SLC7A9 cystinuria patients and carriers: A need for a new
classification. J Am Soc Nephrol 13:2547–2553, 2002.
2. Add 1 mL 5% CTAB in citrate buffer. 11. Nuttall, KL: Porphyrins and disorders of porphyrin metabo-
3. Read turbidity in 5 minutes. lism. In Burtis, CA, and Ashwood, ER: Tietz Fundamentals of
Clinical Chemistry. WB Saunders, Philadelphia, 1996.
12. McKusick, VA, and Neufeld, EF: The mucopolysaccharide storage
diseases. In Stanbury, JB, Wyngaarden, JB, and Fredrickson, DS
(eds): The Metabolic Basis of Inherited Diseases. McGraw-Hill,
TECHNICAL TIP Lesch-Nyhan disease should not be New York, 1983.
13. Kelly, S: Biochemical Methods in Medical Genetics. Charles C.
confused with uromodulin-associated kidney disease, in Thomas, Springfield, IL, 1977.
which the uric acid crystals appear later in life. 14. Garrod, AE: Inborn Errors of Metabolism. Henry Froude &
Hodder & Stoughton, London, 1923.