CHAPTER 9 Urinalysis and Body Fluids Analysis
VII. Metabolic Products in the Urine
A. Homogentisic acid, a product formed in the metabolic pathway of tyrosine, is excreted in the urine of individuals who lack a specific enzyme and develop alkaptonuria. Two screening tests are used to detect homogentisic acid: the ferric chloride test and the silver nitrate test.
1. A transient, dark blue color is seen as two drops of 10% ferric chloride are added to 2 mL urine.
Missing Enzyme Amino Acid Synthetic Products Affected or Metabolite or Metabolic Disorder
Phenylalanine
Phenylalanine hydroxylase X Phenylketonuria (PKU) Catecholamines
Tyrosine Melanin
Thyroxine (T4)
Tyrosine transaminase X Tyrosinemia
Tyrosyluria p-Hydroxyphenylpyruvic acid
p-Hydroxyphenylpyruvate oxidase X Tyrosinemia Tyrosyluria Homogentisic acid
Homogentisic acid oxidase X Alkaptonuria Homogentisic Acid Fumaric and Acetoacetic acid
■ Figure 9–5 Metabolism of phenylalanine and tyrosine.
(Figure courtesy of Barbara Sawyer.)
2. A black color develops after several drops of 10% ammonium hydroxide are added to 0.5 mL urine containing 4 mL of 3% silver nitrate.
B. A second metabolic pathway for tyrosine is responsible for the production of melanin.
When melanin and its precursors are present in urine, the urine turns dark, even black, with exposure to light or air. To distinguish melanin from homogentisic acid, two tests can be performed.
1. In the ferric chloride tube test, a gray or black precipitate will form with melanin.
2. A red color is produced with melanin and sodium nitroprusside.
C. Phenylketonuria (PKU) is a well-known aminoaciduria. It occurs in approximately 1 in every 15,000 births, and most states require that newborn infants be tested for this disease.
If undetected, this condition results in severe mental retardation. Increased amounts of phenylalanine metabolites in urine give it a characteristic mousy odor.
1. PKU is caused by the failure to inherit the gene that produces the enzyme phenylalanine hydrolase. The disease is usually first detected in blood because urinary accumulation of phenylpyruvic acid takes 2 to 6 weeks. A newborn must have adequate ingestion of dietary phenylalanine, which is a major constituent of milk, prior to blood collec- tion. Once detected, dietary restrictions that eliminate phenylalanine are necessary.
Phenylketonuria is diagnosed by measuring plasma amino acids that indicate elevated plasma phenylalanine and phenylalanine/tyrosine ratio.
2. The best known screening test for PKU is the bacterial inhibition test developed by Guthrie.
a. Blood is collected on filter paper disks typically using a heelstick procedure.
b. The blood-impregnated disks are placed on culture media streaked with Bacillus subtilis bacteria.
c. The Guthrie test is sensitive to serum phenylalanine concentrations>4 mg/dL. If increased amounts of phenylalanine are present in the blood, this will counteract the action of an inhibitor present in the media, and Bacillus subtilis will grow around the disk.
3. Phenylpyruvic acid can be detected in urine with the ferric chloride reaction. Phenistix reagent strips are available for the detection of PKU. When dipped into urine containing phenylpyruvic acid, a permanent blue-gray to green-gray color is produced.
D. Other disorders of tyrosine metabolism can result from inherited or metabolic defects. If tyrosine derived from the diet or from the metabolism of phenylalanine is not metabolized, it accumulates in the serum up to 100 times normal, producing tyrosinemia and overflow into the urine (tyrosyluria).
1. Cirrhosis of the liver, renal dysfunction, and rickets are the principal clinical findings in hereditary tyrosinemia, which is rare. More often, transient tyrosinemia, and thus tyrosyluria, occur in low-birth-weight infants and must be distinguished from PKU.
2. Precipitated tyrosine crystals may be seen in urine sediment. A screening test to detect tyrosine in urine uses nitrosonaphthol, which forms red complexes with tyrosine and tyramine, but is nonspecific.
3. Chromatography should be used to confirm the presence of increased levels of tyrosine because normal urine contains some tyrosine.
E. Maple syrup urine disease (MSUD) is one of a group of disorders associated with abnormal branched-chain amino acids.
1. Failure to inherit the gene for the enzyme necessary to produce oxidative decarboxy- lation of the keto acids in the metabolic pathways of leucine, isoleucine, and valine results in their accumulation in the blood and urine. These excess keto acids give urine a characteristic maple syrup odor.
2. The most commonly used screening test for keto acids is the 2,4-dinitrophenylhydrazine (DNPH) reaction. Addition of DNPH to urine containing keto acids produces a yellow turbidity or precipitate.
F. Miscellaneous tests for metabolic products
1. Dysfunction in the metabolism of tryptophan can result in an increase of indican or 5-hydroxyindoleacetic acid (5-HIAA) in the urine.
a. In certain intestinal disorders, including obstruction, the presence of abnormal bacteria, malabsorption syndromes, or a rare inherited disorder (Hartnup disease), increased amounts of tryptophan are converted to indole in the intestine.
b. The excess indole is then reabsorbed into the blood and converted to indican by the liver and excreted into the urine.
c. Indican in urine, when exposed to air, is oxidized to indigo blue.
d. Urinary indican is detected by an acidic ferric chloride solution, which reacts with indican to form a deep-blue or purple color.
2. In another metabolic pathway, tryptophan is converted to serotonin in the argentaffin cells of the intestine.
a. Malignant tumors of the argentaffin cells produce excess amounts of serotonin, which results in elevated levels of the urinary degradation product 5-HIAA.
b. 5-HIAA can be detected in urine with the addition of 1-nitroso-2-naphthol, which produces a purple to black color, depending on the concentration of 5-HIAA.
3. Cystinuria is characterized by defective tubular reabsorption of cystine and the amino acids arginine, lysine, and ornithine after glomerular filtration.
a. The demonstration of multiple amino acids not being reabsorbed rules out the possi- bility of an error in metabolism, although the condition is inherited. Approximately 65% of individuals who have cystinuria tend to form calculi.
b. A fresh, first morning urine specimen should be examined for cystine crystals.
c. A chemical screening test for urinary cystine uses the cyanide-nitroprusside test.
Sodium cyanide reduces cystine, and the free sulfhydryl groups then react with nitroprusside to produce a red-purple color.
4. Homocystinuria is caused by deficiency of the liver enzyme cystathionineβ-synthase.
Homocysteine is rapidly oxidized to homocystine, which accumulates and is excreted in the urine. Children afflicted with this disease may have seizures and thromboses, and they may become mentally retarded.
a. A fresh urine specimen should be tested for homocystine, because it is labile.
b. The cyanide-nitroprusside reaction is positive.
G. Urine calcium
1. Elevated urinary calcium may be seen in individuals who have renal calculi, hyper- parathyroidism, osteoporosis, or multiple myeloma. Occasionally, individuals who take large amounts of calcium supplement might produce high urine calcium. Sulkowitch’s test is a quick qualitative test for increased levels of urinary calcium. Sulkowitch’s reagent consists of oxalic acid, ammonium oxalate, and glacial acetic acid. When re- acted with urinary calcium, calcium oxalate precipitates, producing turbidity that is graded on a scale from 0 to 4.
2. If large amounts of clumped calcium oxalate or calcium carbonate crystals are noted in a freshly voided urine sample, it may be predictive of the presence of renal calculi.
H. Urine porphyrins
1. Porphyrins are intermediate compounds in the production of heme.
2. Porphyrias are a group of disorders resulting from defects in the heme synthesis path- way.
a. Inherited enzyme deficiencies and lead poisoning interrupt the heme synthesis path- way and produce porphyrins, which result from the spontaneous and irreversible oxidation of their respective porphyrinogens. These porphyrins cannot re-enter the heme synthesis pathway and are excreted in urine and stool.
b. Porphyrias are rare disorders. The most common type in North America is porphyria cutanea tarda.
3. The principle circulating porphyrins include uroporphyrin, coproporphyrin, and pro- toporphyrin.
4. Porphyrin precursors commonly found in urine are porphobilinogen and aminole- vulinic acid (ALA).
5. In their oxidative forms, uroporphyrin, coproporphyrin, and protoporphyrin are dark red or purple and fluorescent. The oxidative form of porphobilinogen—porphobilin—
is dark red. Thus, urine containing porphobilinogen and porphobilin may appear dark red, often referred to as “port wine” color.
6. If test results are negative for blood in red-colored urine and the individual is not taking medication that could color the urine, the specimen should be tested for porphyrinuria.
7. There are two screening tests for porphyrinuria: the Ehrlich’s reaction and fluores- cence under ultraviolet light from a Wood’s lamp (see 10 below). In Ehrlich’s reaction, urobilinogen and porphobilinogen react with p-dimethylaminobenzaldehyde (Ehrlich’s reagent) to produce a cherry-red color.
8. To detect ALA, acetylacetone is added to a urine specimen before testing to convert ALA to porphobilinogen. Addition of Ehrlich’s reagent produces a cherry-red color in a specimen that yields positive test results. Increased urinary ALA is a common screening test for lead poisoning.
9. If a cherry-red color is produced by a urine specimen after the addition of Ehrlich’s reagent, the Watson-Schwartz test is performed to differentiate urobilinogen and porphobilinogen based on solubility differences.
a. Chloroform is added to the tube to extract urobilinogen, and porphobilinogen remains in the aqueous phase. The tube is shaken vigorously, and the phases are allowed to separate.
b. If the red color resides only in the chloroform layer, increased amounts of uro- bilinogen are present. If the aqueous layer is red, porphobilinogen or other Ehrlich- reactive substances are present.
10. Fluorescence screening detects the presence of urobilinogen, coproporphyrin, and protoporphyrin. These porphyrins must be extracted into a mixture of glacial acetic acid and ethyl acetate. The solvent layer is then examined with a Wood’s lamp. If the test is positive, the solvent layer fluoresces as pink, violet, or red, depending on the concentration of porphyrins present.
11. The Hoesch screening test for porphobilinogen is a rapid screen for urinary porpho- bilinogen. Two drops of urine are added to the Hoesch reagent, which is Ehrlich’s reagent plus HCl. The uppermost part of the solution turns red in the presence of porphobilinogen.