Primary (metabolic) aminoacidopathies

Phenylketonuria (PKU). Classic PKU is inherited as an autosomal recessive trait. It is uncommon in Asians and African Americans and is due to deficiency of a liver enzyme known as “phenylalanine hydroxylase,” which is needed to convert the amino acid phenylalanine to tyrosine. With its major utilization pathway blocked, phenylalanine accumulates in the blood and leads to early onset of progressive mental deficiency. This disease is one of the more common causes of hereditary mental deficiency and one of the few whose bad effects can be prevented by early treatment of the infant. At birth the infant usually has normal serum levels of phenylalanine (<2 mg/100 ml) due to maternal enzyme activity, although some instances of mental damage in utero occur. In the neonatal period, after beginning a diet containing phenylalanine (e.g., milk), serum phenylalanine levels begin to rise. When the phenylalanine to tyrosine pathway is blocked, some phenylalanine metabolism is shunted to ordinarily little-used systems such as transamination to phenylpyruvic acid.

Urine screening tests. When the serum phenylalanine level reaches 12-15 mg/100 ml, sufficient phenylpyruvic acid is produced that it begins to appear in the urine. This becomes detectable by urine screening tests (Phenistix or the older ferric chloride test) at some time between ages 3 and 6 weeks. Unfortunately, by then some degree of irreversible mental damage may have occurred. Therefore, it is highly desirable to make an earlier diagnosis to begin treatment as soon as possible after birth.

Blood screening tests. The most widely used screening test for elevated serum phenylalanine levels is the Guthrie test. This is a bacterial inhibition procedure. A certain substance that competes with phenylalanine in Bacillus subtilis metabolism is incorporated into culture medium; this essentially provides a phenylalanine-deficient culture medium. Bacillus subtilis spores are seeded into this medium; but to produce significant bacterial growth, a quantity of phenylalanine equivalent to more than normal blood levels must be furnished. Next, a sample of the patient’s blood is added, and the presence of abnormal quantities of serum phenylalanine is reflected by bacterial growth in the area where the specimen was applied. The Guthrie test, if properly done, is adequately sensitive and accurate and reliably detects definitely abnormal levels of serum phenylalanine (і4 mg/100 ml). It also fulfills the requirements for an acceptable screening method. However, there are two controversial aspects to this test. First, the standard practice is to obtain a blood specimen from the infant (usually as a filter paper blood spot using a heel puncture) before discharge from the hospital. In some cases this may result in the specimen being obtained 48 hours or less after birth, with an even shorter duration of phenylalanine intake if milk feeding is not begun soon after birth. There is some controversy in the literature as to whether a significant percentage (about 5%-10%) of infants with PKU will be missed if the specimen is obtained in the first 48 hours of life. A few studies indicate that this is unlikely, but the number of patients was not large enough to conclusively establish this point. Some screening programs recommend second testing of infants discharged before 48 hours of life, and most authorities recommend retesting if the initial test specimen was obtained before 24 hours of life.

Second, PKU is not the only cause of elevated serum phenylalanine levels in the neonate. In fact, more positive (abnormally elevated) Guthrie test results are reportedly caused by non-PKU etiologies than by PKU. A major etiology is temporary (“late enzyme development”) hypertyrosinemia associated with low birth weight, high-protein formulas, and vitamin C deficiency. Some infants with severe liver disease and some with galactosemia have been reported to have elevated serum phenylalanine levels. Since hyperphenylalaninemia is not diagnostic of PKU, and since a long-term low-phenylalanine diet could be harmful to some persons who do not have PKU, an abnormal Guthrie test result should be followed up by more detailed investigation, including, as a minimum, both the serum phenylalanine and tyrosine levels. The typical PKU patient has a serum phenylalanine level greater than 15 mg/100 ml, with a serum tyrosine level less than 5 mg/100 ml. The tests may have to be repeated in 1-2 weeks if values have not reached these levels. DNA probe diagnosis is also available for equivocal or problem cases or prebirth diagnosis.

Phenylketonuria variants. About 10% of infants with apparent PKU have been found to have a PKU variant. The enzyme system for alteration of phenylalanine to tyrosine is actually a group of at least four enzymes and coenzymes. Deficiency in the hydroxylase enzyme produces classic PKU. Deficiency in one of the other components of the system produces variant PKU. In particular, the variant caused by deficiency of the cofactor tetrahydrobiopterin (estimated to account for 0.5%-3.0% of persistent hyperphenylalaninemias) requires therapy in addition to a phenylalanine-deficient diet. Some patients with other variants of PKU do not require a phenylalanine-free diet.

Diagnosis of PKU variants was originally made with a tolerance test using oral phenylalanine (in milk or other materials). Persistence of elevated blood phenylalanine levels greater than 20 mg/100 ml for more than 72 hours was considered indicative of classic PKU, whereas a decrease below the 20-ml level before 72 hours was considered indicative of variant PKU. This association has been challenged by others, and sophisticated tests have been devised to determine the exact etiology of the different forms of persistent hyperphenylalaninemia. Some of these procedures are available only in pediatric research centers. It would seem reasonable to recommend that an abnormal Guthrie test result be immediately followed up with a blood specimen for assay of phenylalanine and tyrosine levels. Afterward, pending results, the infant should be placed on a low-phenylalanine diet. If both phenylalanine and tyrosine levels are high, the patient probably does not have PKU. If only the phenylalanine level is high, and especially if it is greater than 15 mg/100 ml, the infant should be referred to a specialized PKU center for additional studies while the low-phenylalanine diet is continued.

Alkaptonuria (ochronosis). The typical manifestations of this uncommon disease are the triad of arthritis, black pigmentation of cartilage (ochronosis), and excretion of homogentisic acid in the urine. Arthritis usually begins in middle age and typically involves the spine and the large joints. Black pigmentation of cartilage is most apparent in the ears but may be noticed in cartilage elsewhere or may even appear in tendons. The intervertebral disks often become heavily calcified and thus provide a characteristic x-ray picture. The disease is caused by abnormal accumulation of homogentisic acid, an intermediate metabolic product of tyrosine; this, in turn, is caused by a deficiency of the liver enzyme homogentisic acid oxidase, which mediates the further breakdown of the acid. Most of the hemogentisic acid is excreted in the urine, but enough slowly accumulates in cartilage and surrounding tissues to cause the characteristic changes previously described. Diagnosis is established by demonstration of homogentisic acid in the urine. Addition of 10% sodium hydroxide turns the urine black or gray-black. A false positive urine glucose test result is produced by copper reduction methods such as Benedict’s test or Clinitest, whereas glucose oxidase dipstick methods are negative.

Other primary aminoacidopathies. These are numerous, varied, and rare. They are mostly diagnosed by paper chromatography of urine (or serum), looking for abnormal quantities of the particular amino acid involved whose metabolic pathway has been blocked. The most widely known diseases (apart from PKU and alkaptonuria) are maple syrup disease and histidinemia. The most common is homocystinuria. Many of the primary aminoacidopathies can be diagnosed in the first trimester of pregnancy by means of chorionic villus biopsy.

Secondary aminoacidopathies. Secondary aminoacidopathies are associated with a renal defect, usually of reabsorption, rather than a primary defect in the metabolic pathway of the amino acid in question. The serum levels are normal. The most common cause is a systemic disease such as Wilson’s disease, lead poisoning, or Fanconi’s syndrome. In such cases, several amino acids usually are found in the urine. Aminoaciduria may occur normally in the first week of life, especially in premature infants. A much smaller number of patients have a more specific amino acid renal defect with one or more specific amino acids excreted; the most common of these diseases is cystinuria. Patients with cystinuria develop cystine renal calculi. Cystine crystals may be identified in acidified urine, proving the diagnosis. Otherwise, combined urine and serum paper chromatography is the diagnostic method of choice.