Amino Acid Disorders

Phenylalanine-Tyrosine Disorders

Phenylketonuria (PKU)

• It is an autosomal recessive inborn error of metabolism (IEM) that occurs when the gene to produce phenylalanine hydroxylase is not inherited, preventing the conversion of phenylalanine to tyrosine.

• It can cause severe mental retardation, give urine a mousy odor and make skin a fair complexion.

  • Increased amounts of keto acids, including phenylpyruvate, cause a mousy urine odor.

  • The decreased production of tyrosine and its pigmentation metabolite, melanin, causes skin to have a fair complexion.

• It is first diagnosed with blood samples; urine testing is used for confirmation or for monitoring phenylanine levels.

  • The most well-known blood test for PKU is the microbial inhibition assay developed by Robert Guthrie.

    1. Blood collected from the heel is allowed to be absorbed into filter paper circles until completely saturated.

    2. They are then placed on culture media streaked with the bacterium Bacillus subtilis and containing beta-2-thienylalanine, an inhibitor of the bacterium.

    3. If increased phenylalanine levels are present in the blood, phenylalanine counteracts the action of beta-2-thienylalanine and growth will be observed around the paper disks.

  • Urine tests for phenylpyruvic acid are based upon the ferric chloride reaction performed by tube test; however, care must be taken to avoid interferences from other amino acids, medications and diaper powder.

    1. 1 mL of urine is added into a tube.

    2. Five drops of 10% ferric chloride is slowly added.

    3. If phenylpyruvic acid is present, a permanent blue-green color.

  • Dietary changes that eliminate phenylalanine can prevent the excessive buildup of serum phenylalanine.

    • Phenylalanine is a major constituent of milk.

    • As the child matures, alternate pathways of phenylalanine metabolism develop, and dietary restrictions can be eased.

    • Many products that contain large amounts of phenylalanine, such as aspartame, now have warnings for people with PKU.

Tyrosyluria

• It is the increase and accumulation of tyrosine in the plasma that can be inherited or acquired.

  • If inherited, it can present a serious and usually fatal condition that results in both liver and renal tubular disease producing a generalized aminoaciduria.

    • Type 1 is caused by the deficiency of the enzyme fumarylacetoacetate hydrolase (FAH), and it produces a generalized renal tubular disorder and progressive liver failure in infants soon after birth.

    • Type 2 tyrosinemia is the deficiency of the enzyme tyrosine aminotransferase, and it causes corneal erosion and lesions on the palms, fingers and soles of the feet believed to be caused by crystallization of tyrosine in the cells.

    • Type 3 tyrosinemia is the deficiency of the enzyme p-hydroxyphenylpyruvic acid dioxygenase, and it results in mental retardation.

  • If acquired, it is caused by underdevelopment of the liver function required to produce the enzymes necessary to complete the tyrosine metabolism.

    • It seldom results in permanent damage.

    • Tyrosine and leucine crystals may be rarely observed during microscopic examination of the urine sediment.

    • It may be confused with PKU, so it is distinguished by the green color fading rapidly, indicating the presence of tyrosine.

  • It may also be acquired from severe liver disease.

    • It is is a more serious condition.

    • Tyrosine and leucine crystals may be rarely observed during microscopic examination of the urine sediment.

• It is screened using the nitroso-naphthol test; however, it is non-specific and tandem mass spectrophotometry (MS/MS) is used to confirm it.

  1. Five drops of urine are placed in a tube.

  2. 1 mL of 2.63N nitric acid, one drop of 21.5% sodium nitrite and then 0.1 mL of 1-nitroso-2-napthol are added.

  3. The solution is mixed and left to stand for 5 minutes.

  4. The presence of an orange-red color shows a positive reaction

  5. Further testing with MS/MS is needed.

Melanuria

• It is an IEM characterized by the overproliferation of melanocytes, causing malignant melanoma.

  • These tumors secrete a colorless precursor of melanin, 5,6-dihydroxyindole, which oxidizes to melanogen and then to melanin.

  • The melanin appears in the urine and causes it to darken when exposed to air.

• It is screened using ferric chloride, sodium nitroprusside (nitroferricyanide) or Ehrlich reagent.

  • In the ferric chloride tube test, a gray or black precipitate forms in the presence of melanin and is easily differentiated from the reactions produced by other amino acid products.

  • In the sodium nitroprusside test, a red color is produced by the reaction of melanin and sodium nitroprusside.

    • Interference from acetone and creatinine can be avoided by adding glacial acetic acid, which causes melanin to revert to a green-black color, acetone to purple, and creatinine to amber.

Alkaptonuria

• It is an IEM from failure to inherit the gene to produce homogentisic acid oxidase, causing homogentisic acid to accumulate in the body.

• It does not usually manifest in early childhood, but observations of brown-stained or black-stained cloth diapers and reddish-stained disposable diapers have been reported. In later life, brown pigment becomes deposited in the body tissues.

  • It is particularly noticeable in the ears.

  • Deposits in the cartilage eventually lead to arthritis.

  • A high percentage of persons with alkaptonuria develop liver and cardiac disorders.

• Urine from patients darken after becoming alkaline from standing at room temperature.

• It is screened using the ferric chloride test, Clinitest, the homogentistic acid test, through spectrophotometry or through chromatography.

  • In the ferric chloride test, a transient deep blue color is produced if it is present.

  • In the Clinitest, a yellow precipitate is produced if it is present.

  • In the homogentistic acid test, a black color is produced if it is present; however, it can be interfered by large amounts of ascorbic acid.

    1. 4 mL of 3% silver nitrate is placed in a tube.

    2. 0.5 mL of urine is added and the solution is mixed.

    3. 10% NH4OH is then added by drops until a black color is observed.

  • Spectrophotometry and chromatograpy are used to to obtain quantitative measurements of both urine and plasma homogentisic acid.

Branched-Chain Amino Acid Disorders

Maple Syrup Urine Disease (MSUD)

• It is an IEM inherited as an autosomal recessive trait that prevents oxidative decarboxylation of keto acids and causes their accumulation in the body.

• Newborns with MSUD begin to exhibit failure to thrive after approximately l week, progressing into severe mental retardation and death.

• An indicator is a strong maple syrup odor in the urine, and it is then confirmed and monitored by screening with the 2,4-dinitrophenylhydrazine (DNPH) reaction; however, it is not specific.

  1. 1 mL of urine is added to a tube.

  2. 10 drops of 0.2% 2,4-DNPH in 2N HCl are then added.

  3. The solution is left to stand for 10 minutes.

  4. A yellow or white precipitate must be seen in the presence of keto acids.

• If detected by the 11th day, dietary regulation and careful monitoring of urinary keto acid concentrations can control the disorder.

Organic Acidemias

• These may be isovaleric, propionic and methylmalonic.

  • Isovaleric acidemia is caused by the accumulation of isovalerylglycine due to a deficiency of isovaleryl coenzyme A in the leucine pathway.

  • Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting isoleucine, valine, threonine, and methionine to succinyl coenzyme A.

    • Propionic acid is the immediate precursor to methylmalonic acid in this pathway.

• Generalized symptoms of the organic acidemias include early severe illness with vomiting and metabolic acidosis, hypoglycemia, ketonuria and increased serum ammonia.

  • Isovaleric acidemia is characterized by urine specimens, and sometimes even the patient, having a characteristic odor of “sweaty feet.”

• There is no urine screening test for isovaleric and propionic acidemia, but methylmalonic aciduria can be screened using ρ-nitroaniline.

  1. One drop of urine is placed in a tube.

  2. 15 drops of 0.1% p-nitroaniline in 0.16 M HCl and 5 drops of 0.5% sodium nitrite are then added in order.

  3. The solution is mixed.

  4. 1 mL of 1 M sodium acetate buffer at pH 4.3 is then added.

  5. The solution is boiled for 1 minute.

  6. Five drops of 8N NaOH are then added.

  7. An emerald green is observed if methylmalonic acid is present.

Tryptophan Disorders

Indicanuria

• In cases of intestinal disorders, increased amounts of tryptophan from food are converted to indole, which are then reabsorbed and circulated to the liver, where it is converted to indican and then excreted in the urine.

  • Indican excreted in the urine is colorless until oxidized to the dye indigo blue by exposure to air.

• Early diagnosis is sometimes made when mothers report a blue staining of their infant’s diapers, referred to as the “blue diaper syndrome.”

  • Urinary indican reacts with acidic ferric chloride to form a deep blue or violet color that can subsequently be extracted into chloroform.

• It is screened with the ferric chloride test, and it forms a deep blue or violet color that can subsequently be extracted into chloroform.

• Correction of the underlying intestinal disorder returns urinary indican levels to normal except for cases of Hartnup disease.

  • It affects not only the intestinal reabsorption of tryptophan but also the renal tubular reabsorption of other amino acids, resulting in a generalized aminoaciduria (Fanconi syndrome).

    • The defective renal transport of amino acids does not appear to affect other renal tubular functions.

  • Hartnup disease is treated with dietary supplements and niacin.

5-Hydroxyindoleacetic Acid (5-HIAA)

• It occurs when when carcinoid tumors involving argentaffin (enterochromaffin) cells develop, producing excess amounts of serotonin that degreade into 5-HIAA.

  • Serotonin is produced from tryptophan by the argentaffin cells and is normally used up with only small amounts of its degradation product, 5-HIAA, excreted in the urine.

  • The normal daily excretion of 5-HIAA is 2 to 8 mg, and excretion of greater than 25 mg/24 h can be an indication of argentaffin cell tumors.

• It is screened using nitrous acid and 1-nitroso-2-naphthol, and the appearance of a purple to black color, depending on the amount of 5-HIAA present, indicates a positive result.

  • The test can be performed on a random or first morning specimen; however, false-negative results can occur based on the specimen concentration and also because 5-HIAA may not be produced at a constant rate throughout the day.

  • If a 24-hour sample is used, it must be preserved with hydrochloric or boric acid.

  • Patients must be given explicit dietary restrictions to foods such as bananas, pineapples and tomatoes, and medications such as phenothiazines and acetanilids for 72 hours prior to specimen collection.

Cystine Disorders

Cystinuria

• It is an inherited disorder that makes the renal tubules unable to reabsorb cystine filtered by the glomerulus, though its severity depends on the type of inheritance.

  • One type is unable to reabsorb all four amino acids - cystine, lysine, arginine and ornithine - and patients are more prone to producing renal calculi early in life (65%).

  • Another type is unable to absorb only cystine and lysine, and patients are less likely to produce renal calculi.

• Physical screening is based on the observation of cystine crystals in the sediment of concentrated or first morning specimens.

• Chemical screening makes use of cyanide-nitroprusside; however, false-positive results may occur in the presence of ketones and homocystine.

  1. 3 mL of urine is placed in a tube.

  2. 2 mL of sodium cyanide is then added.

  3. The solution is left to stand for 10 minutes.

  4. Five drops of 5% sodium nitroprusside are then added.

  5. A red-purple color is observed if cystine is present.

Cystinosis

• It is an IEM that causes a defect in the lysosomal membranes, preventing the breakdown of cystine which produces crystalline deposits in many areas of the body.

  • The renal tubules, particularly the proximal convoluted tubules, are affected by the deposits of cystine that interfere with reabsorption and Fanconi syndrome occurs.

• It can be categorized as nephopathic (subdivided into infantile and late-onset) or non-nephropathic.

  • In infantile nephropathic cystinosis, there is rapid progression to renal failure.

  • In late-onset nephropathic cystinosis there is a gradual progression to total renal failure.

  • Non-nephropathic cystinosis is relatively benign but may cause some ocular disorders.

• Routine laboratory findings in infantile nephropathic cystinosis include polyuria, generalized aminoaciduria, positive test results for reducing substances, and lack of urinary concentration.

• Continued deposition of cystine results in renal failure early in life, and it can become a benign form and be fatal.

• Renal transplants and the use of cystine-depleting medications to prevent the buildup of cystine in other tissues extends the patient’s lifespan.

Homocystinuria

• It is caused by defects in the metabolism of the amino acid methioine, producing an increase in homocystine throughout the body.

• It can result in failure to thrive, cataracts, mental retardation, thromboembolic problems, and death.

• Screening is done with the cyanide-nitroprusside test confirmed with a silver-nitroprusside test, or it can be done with MS/MS.

  • 1 mL of urine is placed in a tube.

  • Two drops of concentrated NH4OH and 0.5 mL of 5% silver nitrate are then added.

  • The solution is left to stand for 10 minutes.

  • Five drops of sodium nitroprusside are then added.

  • A red-purple color is observed if homocystine is present.

• Early screening for newborns and a change in diet can alleviate metabolic problems.

Porphyrin Disorders

• Porphyrins are the intermediate compounds in the production of heme.

• The solubility of porphyrin compounds varies with their structure.

  • ALA (α-aminolevulinic acid), porphobilinogen, and uroporphyrin are the most soluble and readily appear in the urine.

  • Coproporphyrin is less soluble but is found in the bile, feces and urine.

  • Protoporphyrin is not seen in the urine but is found in the bile, blood and feces.

• Porphyrin disorders can be acquired or inherited from erythrocytic and hepatic malfunctions or exposure to toxic agents.

  • Common causes of acquired porphyrias include lead poisoning, excessive alcohol exposure, iron deficiency, chronic liver disease, and renal disease.

  • Inherited porphyrias are much rarer than acquired porphyrias and are caused by failure to inherit the gene that produces an enzyme needed in the metabolic pathway.

    • The inherited porphyrias are frequently classified by their clinical symptoms, either neurologic/psychiatric or cutaneous photosensitivity or a combination of both.

• An indication of the possible presence of porphyrinuria is the observation of a red or port wine color to the urine after exposure to air.

  • The port wine urine color is more prevalent in the erythropoetic porphyrias, and staining of the teeth may also occur.

  • The presence of congenital porphyria is sometimes suspected from a red discoloration of an infant’s diapers.

• Increased porphobilinogen is associated with acute intermittent porphyria.

• The two screening tests for porphyrinuria use the Ehrlich reaction and fluorescence under ultraviolet light in the 550 to 600 nm range.

  • The Ehrlich reaction can be used only for the detection of ALA and porphobilinogen.

    • Acetylacetone must be added to the specimen to convert the ALA to porphobilinogen prior to performing the Ehrlich test.

    • The Ehrlich reaction, including the Watson-Schwartz test for differentiation between the presence of urobilinogen and porphobilinogen and the Hoesch test, were discussed in detail in Chapter 5.

    • Testing for the presence of porphobilinogen is most useful when patients exhibit symptoms of an acute attack.

  • The fluorescent technique must be used for the other porphyrins.

    • It makes use of extraction into a mixture of glacial acetic acid and ethyl acetate and the examination of the resulting solvent layer.

    • Negative reactions have a faint blue fluorescence while positive reactions have violet, pink or red, depending on the concentration of porphyrins.

    • If the presence of interfering substances is suspected, the organic layer can be removed to a separate tube and 0.5 mL of hydrochloric acid is added to the tube.

      • Only porphyrins are extracted into the acid layer, which then produces a bright orange-red fluorescence.

    • The fluorescence method does not distinguish among uroporphyrin, coproporphyrin and protoporphyrin, but it rules out porphobilinogen and ALA.

  • The identification of the specific porphyrins requires additional techniques and the analysis of fecal and erythrocyte samples.

  • Increased protoporphyrin is best measured in whole blood.

    • Testing for the presence of porphobilinogen is most useful when patients exhibit symptoms of an acute attack.

    • A negative test result is obtained in the presence of lead poisoning unless ALA is first converted to porphobilinogen.

Mucopolysaccharide Disorders

• Mucopolysaccharides, or glycosaminoglycans, are a group of large compounds located primarily in the connective tissue consisting of a protein core with numerous polysaccharide branches.

• The products most frequently found in the urine are dermatan sulfate, keratan sulfate and heparan sulfate, with the appearance of a particular substance being determined by the specific metabolic error that was inherited.

• There are many types of mucopolysaccharidoses, but the best known are Hurler syndrome, Hunter syndrome, and Sanfilippo syndrome.

  • In Hurler syndrome, mucopolysaccharides accumulate in the cornea of the eye, the skeletal structure is abnormal and there is severe mental retardation.

    • It is usually fatal during childhood.

  • In Hunter syndrome, the skeletal structure is abnormal and there is severe mental retardation.

    • It is a sex-linked recessive disorder and is rarely seen in females.

    • It is usually fatal during childhood.

  • In Sanfilippo syndrome, the only abnormality is mental retardation.

• The most frequently used screening tests are the acid-albumin and cetyltrimethylammonium bromide (CTAB) turbidity tests and the metachromatic staining spot tests.

  • In both the acid-albumin and the CTAB tests, a thick, white turbidity forms when these reagents are added to urine that contains mucopolysaccharides.

    1. 5 mL of urine is placed in a tube.

    2. 1 mL of 5% CTAB in citrate buffer is then added.

    3. After 5 minutes, the turbidity is examined and graded on a scale of 0 to 4.

  • Metachromatic staining procedures use basic dyes to react with the acidic mucopolysaccharides.

    1. Filter paper is dipped into 0.59% azure A dye in 2% acetic acid and allowed to dry.

    2. One drop of urine is then added to the paper.

    3. The paper is washed with 1 mL acetic acid + 200 mL methanol diluted to a liter.

    4. If mucopolysaccarides are present, a blue color will be observed.

• Bone marrow transplants and gene replacement therapy are the most promising treatments for these disorders.

Purine Disorders

• The main purine disorder is Lesch-Nyhan disease, which is a sex-linked recessive disease that makes it unable to produce the enzyme hypoxanthine guanine phosphoribosyltransferase, causing the accumulation of uric acid throughout the body.

• Patients suffer from severe motor defects, mental retardation, a tendency toward self-destruction, gout and renal calculi.

• Development is usually normal for the first 6 to 8 months, with the first symptom often being the observation of uric acid crystals resembling orange sand in diapers.

• Laboratories should be alert for the presence of increased uric acid crystals in pediatric urine specimens.

Carbohydrate Disorders

• The presence of increased urinary sugar (melituria) is most frequently due to an inherited disorder; however, the majority of meliturias cause no disturbance to body metabolism.

  • However, a melituria of concern is galactosuria, indicating the inability to properly metabolize galactose to glucose.

    • The resulting galactosemia with toxic intermediate metabolic products results in infant failure to thrive, combined with liver disorders, cataracts, and severe mental retardation.

    • Galactosuria can be caused by a deficiency in any of three enzymes, galactose-1-phosphate uridyl transferase (GALT), galactokinase and UDP-galactose-4-epimerase.

      • Of these enzymes, it is GALT deficiency that causes the severe, possible fatal symptoms associated with galactosemia.

      • Galactose kinase deficiency can result in cataracts in adulthood.

      • UDP-galactose-4-epimerase deficiency may be asymptomatic or produce mild symptoms.

    • Early detection of galactosuria followed by removal of lactose (a disaccharide containing galactose and glucose ) from the diet can prevent these symptoms.

  • Lactosuria may be seen during pregnancy and lactation.

  • Fructosuria is associated with parenteral feeding and pentosuria with ingestion of large amounts of fruit.

• Additional tests including chromatography can be used to identify other non-glucose reducing substances.