Vol.24, pp. 301-309,1991 Printedin Canada.All rightsreserved.
0009-9120/91$3.00 + .00 Copyright©1991The CanadianSocietyof Clinical Chemists.
Clin Biochem,
Detection of Hereditary Metabolic Disorders Involving Amino Acids and Organic Acids VIVIAN E. SHIH Amino Acid Disorder Laboratory, Massachusetts General Hospital, Boston, MA, USA, and Department of Neurology, Harvard Medical School, Boston, MA, USA Inherited disorders in the metabolism of amino acids and organic acids may cause neurological dysfunction and acute metabolic crises. For many of these disorders, early diagnosis and early treatment can greatly improve the outcome. A general description of the clinical manifestations and a discussion of selected techniques and approaches for the laboratory diagnosis are reviewed.
ratory diagnosis. Readers are referred to several excellent publications which include detailed descriptions of both the clinical and biochemical findings (2-4) as well as the laboratory techniques (5-7).
KEY WORDS: inborn errors of metabolism; amino acids; laboratory diagnosis.
Clinical m a n i f e s t a t i o n s
Introduction
n the past three decades, the clinical applicaIresulted tion of analytical biochemical techniques has in the discovery of many inborn errors of amino acid and organic acid metabolism. At least 150 disorders are now known and together their frequency is estimated to be in the order of I in 2,000 to 1 in 3,000 newborns (1). These metabolic disorders are of clinical importance because many cause severe clinical diseases. Many also respond well to treatment. Laboratory detection of inborn errors of amino and organic acid metabolism is now available at various medical centers and commercial laboratories. Screening for these disorders is carried out by various chromatographic techniques to detect the changes in metabolite pattern resulting from an enzyme block. Due to the rarity and complexity of some of these metabolic disorders, the referring clinician often relies upon the clinical biochemist to interpret the laboratory findings and advise on further diagnostic workup and management. It is now standard practice to include such interpretations when reporting results of metabolic screening. A general description of the clinical manifestations of the disorders of amino acid and organic acid metabolism will be followed by a discussion of selected techniques and approaches for their labo-
Correspondence: Vivian E. Shih, Massachusetts General Hospital, Amino Acid Laboratory, Bldg. 149, 13th Street, Boston, MA 02129, USA. M a n u s c r i p t received December 21, 1990; revised March 4, 1991; accepted April 24, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
Disorders of amino acid and organic acid metabolism are, with a few exceptions, autosomal recessively inherited traits. The carrier parents are usually normal and, with each pregnancy, there is a 1-in-4 chance of an affected child. The clinical manifestations of these metabblic disorders vary considerably from neonatal death to mild neuropsychiatric symptoms in adulthood. However, the majority of patients come to medical attention in infancy or childhood. Many of these disorders affect the central nervous system, and the most common and nonspecific finding is developmental delay. Hypotonia, myoclonic seizures and choreoathetoid movements have been observed in a number of metabolic disorders, particularly those involving organic acids. Spastic paraparesis and awkward gait can be late complications of hyperammonemia. Infection often precipitates episodes of vomiting, ataxia and lethargy with progression to coma in patients with a number of amino acid and organic acid disorders. Such acute metabolic encephalopathy due to hyperammonemia or metabolic ketoacidosis is a life-threatening complication. Other clinical features suggesting the need for metabolic screening include dislocated optical lenses, unexplained hepatomegaly or cardiomyopathy, and vascular occlusion. Symptoms of acute pancreatitis in children have recently been seen in association with several metabolic disorders (methylmalonic acidemia, maple syrup urine disease, isovaleric acidemia, and homocystinuria) (8). Mild facial dysmorphism and anomalies such as cystic kidney have now been described in several organic disorders, including lactic acidosis, glutaric acidemia Type II, and cobalamin F mutant (2,9). Macrocephaly is a prominent sign in N-acetylaspartic aciduria associated with Canavan's disease. 301
SHIH TABLE 1
Simple General Urine Screening Tests Inspection for color and odor Dinitrophenylhydrazine (DNPH) and/or FeC13 test for a-keto acids Cyanide-nitroprusside test for disulfides Test for reducing substances Test for ketones Dipstick for sulfite
It is notable that not all amino acidurias cause clinical disease. Many of the following disorders have been detected in asymptomatic individuals by routine newborn screening and family survey: histidinemia, hyperprolinemia type 1, cystathioninemia, hyperlysinemia, Hartnup disorder, familial renal iminoglycinuria and dicarboxylic aminoaciduria. Early diagnosis is important as early treatment can prevent brain damage in many of these disorders. Long-term management includes nutritional therapy and/or megavitamin administration. Acute metabolic decompensation associated with hyperammonemia or metabolic ketoacidosis requires aggressive treatment including peritoneal dialysis or hemodialysis. Organ transplant is a promising new form of enzyme therapy.
Laboratory detection Specimen collection is a very important step in the detection of metabolic disorders. In acutely ill patients, the blood and urine specimens on admission are most appropriate for metabolic screening and should be saved from all patients whose diagnosis is unclear. Blood chemistry such as gases, pH, electrolytes, anion gap, glucose and ammonia provides clues to the nature of the metabolic disorder. These data as well as a brief clinical resume should be made available to the biochemist for interpretation of the screening results. Routine metabolic screening usually includes several simple preliminary tests and chromatographic analysis of amino acids and organic acids. Serum carnitine levels and biotinidase screening are useful ancillary tests. Other more specific tests depend on the clinical findings and initial test results. PRELIMINARY GENERAL SCREENING TESTS
All urine specimens for metabolic screening are subjected to a battery of simple general screening tests (Table 1). Inspection of urine for color and odor often gives important clues to a metabolic derangement. For example, characteristic odors are found in maple syrup urine disease (MSUD) and sweaty feet syndrome (isovaleric acidemia) which
302
are disorders of leucine metabolism, and fish odor syndrome which is due to the accumulation of trimethylamine. The dinitrophenylhydrazine (DNPH) and ferric chloride tests detect alpha-ketoacids in disorders such as phenylketonuria (PKU), MSUD, and tyrosinemia I. The cyanide nitroprusside test detects disulfides such as cystine, homocystine and 3-mercaptolactate-cysteine mixed disulfide. Medications containing sulfhydryl group such as captopril, penicillamine, dimercaptosuccinic acid, and N-acetylcysteine react with the nitroprusside reagent and interfere with this test. The presence of reducing substances and ketone bodies in urine can be detected by commercially available reagent tablets and test strips. Sulfite screening should be performed only on a fresh urine sample, and preferably in combination with thiosulfate determination, to detect molybdenum cofactor deficiency/ sulfite oxidase deficiency. A M I N O ACID ANALYSIS
Urine amino acid screening is performed in our laboratory using high voltage electrophoresis at pH 1.9 followed by paper chromatography in butanol-acetic acid-water (12:3:5) for two-dimensional separation (5). Thin-layer chromatography is an alternative method. Quantitative analysis is used by some laboratories for screening. Blood amino acids are quantified on an amino acid analyzer or a high-performance liquid chromatography (HPLC) system. The technique should be sensitive enough to measure amino acid concentrations as low as 3 ~moles/L. This limit is important because a low citrulline level is a diagnostic criterion of defects in urea synthesis. Free argininosuccinate in plasma or amniotic fluid can be difficult to detect and quantify since it often coelutes with another compound. Therefore, the sample should be boiled in acid before analysis to convert the free acid to its stable anhydrides. In normal individuals, circulating homocysteine, which has a sulphydryl group, is bound to serum proteins. In pathological conditions, the bound homocysteine is likely to be increased before the free disulphide form, i.e., homocystine, accumulates. Thus, measurement of total (free + bound) homocysteine rather than only free homocystine is a more sensitive diagnostic test for mild homocyst(e)inemia. Several methods have been developed for quantifying total homocysteine and cysteine, and all involve reduction and release of these protein-bound amino acids by a thiol reagent before analysis (10:12). ORGANIC ACID ANALYSIS
Gas chromatography using capillary column in combination with mass spectrometry (GC/MS) is
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
HEREDITARY METABOLIC DISORDERS
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Figure 1--Comparison of urine organic acid profiles in medium-chain acyl-CoA dehydrogenase deficiency (MCAD DEF., top) and consumption of dietary medium-chain triglycerides (bottom). the preferred technique for organic acid analysis. Table-top models with automated computerized analysis of the mass spectra are now available at affordable prices. Organic acids are isolated from blood and urine by solvent extraction, anion exchange column, or batch isolation using silicic acid before derivatization to the trimethylsilyl or methyl esters. Oxime formation is useful for the detection of ketoacids, aldehydes and ketones. Quantification of organic acids is notoriously inaccurate, largely due to inconsistent efficiency of extraction. Hoffmann et al. (13) recently described an improved method for sample preparation suitable for quantitative analysis. Measurements using selected ion monitoring and stable isotope dilution are sensitive and accurate techniques (14) which are applicable to prenatal diagnosis of organic acid disorders. Screening for volatile short chain fatty acids is not the test of choice for detection of the short chain fatty acid disorders such as isovaleric aci-
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
demia or propionic acidemia. The volatile fatty acids are often not increased during remission and the diagnosis can best be made by detecting their nonvolatile glycine conjugates (e.g., isovalerylglycine) and other characteristic metabolites (e.g., methylcitrate or 3-hydroxypropionate) which are present in blood and urine at all times. CARNITINE MEASUREMENT
Carnitine plays an important role in transporting fatty acids into and out of mitochondria where their oxidation takes place. Total serum carnitine consists of two fractions, free and esterified (5,15). A lower than normal ratio of the free-to-esterified serum carnitine signifies the presence of excessive organic acids. The total serum carnitine may be decreased in organic acidurias. Slightly reduced serum free carnitine is common in patients on valproate treatment or on a ketogenic diet. Analysis of acylcarnitines in the urine by fast atom born-
303
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Figure 2--Evolution of urine organic acid pattern during acute metabolic decompensation in propionic acidemia. Top, on admission; middle, after one day of treatment; bottom, after three days of treatment.
304
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
HEREDITARY METABOLIC DISORDERS TABLE 2 Differential Diagnosis of Urea Cycle Disorders Blood Plasma Diagnostic Urine Ammonia Citrulline Amino Acids Orotic Acid
Deficiency N-acetylglutamate synthetase Carbamylphosphate synthetase Ornithine transcarbamylase Argininosuccinate (ASA) synthetase ASA lyase Arginase
1'
$
-
normal
T
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-
normal
1'
~
-
1`
1`
1' 1'
citrulline
1'
1` 1'
1` N
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1' 1'
bardment (FAB) mass spectrometry (16,17) has
impregnated on filter paper. The frequency of biotinidase deficiency, as detected by newborn screening, is approximately one in 60,000, half with profound deficiency (20). Since biotinidase deficiency can result in significant clinical disability, for which simple and effective t r e a t m e n t is available, screening for this disorder among children with unexplained multisystem problems is justified.
b e e n a useful t e c h n i q u e in the diagnosis of a n u m ber of organic acid disorders. BIOTINIDASE SCREENING
Biotin is a vitamin cofactor for four carboxylases. Biotinidase hydrolyzes biotin from biotinyl peptides and biocytin, and is responsible for the recycling of biotin. Deficient biotinidase activity results in biotin deficiency. Many clinical manifestations are associated with biotinidase deficiency including seizures, mental retardation, skin rashes, vision and hearing impairment, loss of hair, as well as an organic aciduria (18). Heard and colleagues (19) have developed a semiquantitative screening test for biotinidase activity which requires only a drop of serum or blood
OROTIC ACIDMEASUREMENT
U r i n e orotic acid e x c r e t i o n is a v a l u a b l e test in the differential diagnosis of h y p e r a m m o n e m i c syndromes. T h e r e are t h r e e m e t h o d s for its m e a s u r e ment. In both t h e colorimetric m e a s u r e m e n t described by K e s n e r et al. (21) and the q u a n t i t a tive GC/MS m e t h o d r e c e n t l y described by Hoffm a n n et al. (13), sample p r e p a r a t i o n is by liquid
TABLE 3 Differential Diagnosis of Homocystinuria Urine
Plasma Methionine Cystathionine synthase deficiency
Methylation Defects Folate disorders Cobalamin disorders* cblC, cblD, cblF cblE, cblG
T
Urine Cystathionine
Methylmalonate
normal
normal
normal or ~
normal or 1'
normal
normal or ~ normal or $
normal or 1' normal
1' normal
*Eight genetic cobalamin mutants have been described: cblA and cblB affect only methylmalonate metabolism; cblC, cblD, and cblF affect both homocysteine and methylmalonate metabolism; cblE and cblG affect only homocysteine metabolism.
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
305
SHIH TABLE 4 Causes of Neonatal Hyperphenylalaninemia Classic phenylketonuria Pterin cofacter disorders Mild persistent hyperphenylalaninemia Transient hyperphenylalaninemia Hereditary tyrosinemias Neonatal transient tyrosinemia Maternal phenylketonuria
ornithine transcarbamylase deficiency. An HPLC method described by Brusilow and H a u s e r (22) has the advantage of separating orotic acid and orotidine on a n anion exchange column. The elution time of these compounds is easily altered by column conditions, and proper identification of the peaks is essential.
Interpretation and discussion NORMALVARIATIONAND EFFECTSOF DIET, MEDICATION AND TREATMENT
partition chromatography on silicic acid. This, however, does not extract orotidine which is an important metabolite to monitor in carrier testing for
For proper interpretation of screening results it is important to recognize the normal variation of
TABLE 5 Metabolic Disorders Detected Over a 24-Month Period in Author's Laboratory
By Amino Acid Analysis Ornithine transcarbamylase deficiency1 Nonketotic hyperglycinemia Ornithine ~minotransferase deficiency (gyrate atrophy) Argininosuccinate lyase deficiency Carbamylphosphate synthetase deficiency1 Argininosuccinate synthase deficiency Arginase deficiency HHH (hyperornithinemia, hyperammonemia, homocitrullinuria) syndrome Homocystinuria (cystathionine ~-synthase deficiency) Hyperprolinemia type I Mild hyperphenylalaninemia Vitamin B12 disorder (cblC) Cystinuria Total
1 1 1 1 29
By Organic Acid Analysis Medium-chain acyl-CoA dehydrogenase deficiency Methylmalonic acidemia Propionic acidemia Glutaric acidemia type I (glutaryl CoA dehydrogenase deficiency) Glutaric acidemia type II Biotinidase deficiency 2-Methylacetoacetyl CoA thiolase deficiency 4-Hydroxybutyric acidemia (succinic semialdehyde dehydrogenase deficiency) Fumarase deficiency Ethylmalonic acidemia N-acetylaspartic aciduria Short bowel syndrome 2 Ethylene glycol poisoning2 Total
1 1 1 1 1 19
1Suspected by amino acid and orotic acid analysis and confirmed by enzyme assay. 2Acquired abnormalities.
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CLINICAL BIOCHEMISTRY,VOLUME 24, AUGUST 1991
HEREDITARY METABOLIC DISORDERS TABLE 6
Inherited Metabolic Disorders Detected in Sudden Infant Death (SID) Fatty acid oxidation defects Urea cycle disorders Maple syrup urine diseases Biotinidase deficiency Nonketotic hyperglycinemia Vitamin B12 disorder (cblF) Glycogenesis defects Systemic carnitine deficiency 3-Hydroxy-methylglutaric aciduria Pyruvate dehydrogenase deficiency
the urinary amino acid pattern on the basis of age, diet and medication. Infants less than six months of age normally excrete large amounts of proline, hydroxyproline, and glycine, but this pattern is abnormal when present in older individuals. Homocitrulline in the urine of infant is usually from infant formula and is only rarely due to a metabolic disorder such as the hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome or hyperlysinemia. Urinary glycine is quite variable and is also affected by diet (e.g., gelatin) and medication (e.g., valproate). Persistent hyperglycinuria suggests carrier status for familial renal iminoglycinuria and is rarely associated with other hereditary syndromes such as autosomal dominant glucoglycinuria and X-linked hypophosphatasia (2). Organic acidopathy should always be ruled out as the cause of glycine accumulation. If nonketotic hyperglycinemia is suspected, the diagnosis should be confirmed by a decreased plasma/ CSF glycine ratio. Chalmers and Lawson have compiled extensive data on the urine organic acid pattern in health and disease (23). Neonates excrete larger quantities of ethylmalonate, alpha-ketoglutarate, and intermediary compounds of the critic acid cycle than do older infants or children. Organic aciduria of dietary origin can interfere with the interpretation of organic acid analysis. As illustrated in Figure 1, the C6-Clo dicarboxylic acids are increased in the urine from a normal infant fed a formula containing medium-chain triglycerides (MCT), as well as in the urine from a patient with medium chain acyl CoA dehydrogenase deficiency (MCADD). The diagnostic metabolites for this fatty acid oxidation defect are the acylglycine (14) or the acylcarnitine (16,17) derivatives of the dicarboxylic acids. Phenylpropionate is a product of intestinal flora and, when absorbed, is metabolized by mediumchain acyl CoA dehydrogenase. In patients with MCADD, this compound and its glycine conjugate can be found in the urine. Monitoring phenylpropionylglycine excretion following oral administration of phenylpropionate has been used as an aid in diagnosing MCADD (14,24).
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
Metabolites of valproate, a popular anticonvulsant drug, are frequently encountered in the urine and can mask an underlying organic acid disorder if organic acid screening is performed without the use of mass spectrometry. An example of how the timing of sample collection changes the urine organic and acid pattern is illustrated in a patient with propionic acidemia admitted in acute metabolic decompensation (Figure 2). The top panel, on admission, shows large quantities of ketones as well as 3-hydroxyvalerate and 3-hydroxypropionate, a pattern often seen during metabolic decompensation in propionic acidemia. After one day of intravenous fluid and electrolyte therapy, the ketones disappeared and methylcitrate and tiglylglycine were prominent (middle panel); after 3 days, only a few organic acids (lower panel) were detected. The diagnosis could have been difficult if this last sample were the only one analyzed. DIFFERENTIAL DIAGNOSIS
Hyperammonemia, hypoglycemia and ketonuria often accompany organic acid disorders, and their presence is valuable in the differential diagnosis of these disorders. The blood ammonia level in propionic acidemia and methylmalonic acidemia can be as high as that in urea cycle disorders. Mild elevation of blood ammonia together with hypoglycemia and low-ketone production is characteristic of defects in fatty acid oxidation. Recurrent severe ketosis suggests a ketolytic defect due to ketothiolase deficiencies. Lactic acidemia/aciduria is common in children. Among the many causes are impaired gluconeogenesis, defects in pyruvate metabolism, mitochondrial diseases, and any cause of hypoxia or hypoperfusion. In patients presenting with an acute encephalitic picture and/or hyperammonemia, urea cycle enzymopathies should always be considered. Ornithine transcarbamylase deficiency, in particular, is relatively common. The differential diagnosis of urea cycle disorders is shown in Table 2. Although hyperammonemia is a prominent sign in these disorders, the blood ammonia level can be normal during remission and high only after meals. The diagnosis of argininosuccinate synthetase deficiency, argininosuccinate lyase deficiency, and arginase deficiency can easily be made by finding their respective characteristic amino acid abnormalities. In contrast, a low plasma citrulline level may be the only clue to deficiencies of N-acetylglutamate synthetase, carbamylphosphate synthetase, and ornithine transcarbamylase. The two synthetase deficiencies can be positively identified only by enzyme measurement, whereas the combination of hyperammonemia, hypocitrullinemia, and orotic aciduria almost always indicates orni-
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SHIH
thine transcarbamylase deficiency. Female heterozygotes of this X-linked disorder may be normal, mildly affected, or severely affected, depending on the random inactivation of the X-chromosome (Lyonization). Asymptomatic individuals can be identified by the allopurinol test (25), the protein loading test (26), or DNA analysis (26). Homocystinuria is a manifestation of at least eight inborn errors of metabolism (Table 3). Cystathionine ~-synthase deficiency is the most frequently seen and is often associated with a high methionine level. In contrast, in methylation defects the methionine level is normal or low, but cystathionine and methylmalonate may be slightly increased. The total homocystine level is often increased in acquired vitamin Ble and folate deficiencies (12) and as a side effect of medications such as methotrexate, 6-azauridine, and isonicotinic acid hydrazide. Protein-bound homocysteine has also been considered a possible risk factor for arteriosclerotic disease (11). Pipecolic acid, formed in the metabolism of lysine, is primarily metabolized by peroxisomes, and its presence is an indication of a derangement of peroxisomal metabolism (27). Newborn screening for P K U is mandated by law in the United States. Hyperphenylalaninemia in the newborn period is not a single entity. As listed in Table 4, classic P K U is one of seven types of hyperphenylalaninemia. All neonates with hyperphenylalaninemia should have a pterin screening (28) for cofactor deficiency. Increased blood phenylalanine can be due to benign persistent mild hyperphenylalaninemia, may be secondary to neonatal tyrosinemia, or may be a transient finding. Infants of a P K U woman untreated during pregnancy will have increased blood phenylalanine during the first 24 h of life. DISORDER DETECTED
Our laboratory receives blood and urine specimens for metabolic screening from hospitals in the Boston area and several neighboring states. During the past 24 months, we have performed approximately 4,800 tests for the detection of disorders of amino acid and organic acid metabolism. These samples originated from a selected high-risk group, and multiple tests were frequently ordered on the same patient. The disorders detected are listed in Table 5. In addition to these, there were 6 cases of renal Fanconi syndrome, 1 case of trimethylaminuria (fish odor syndrome), 35 cases of lactic aciduria and at least 48 cases of ketonuria with unconfirmed underlying defects. P R E N A T A L A N D P O S T M O R T E M DIAGNOSIS
Blood cells and cultured skin fibroblasts and lymphoblasts are convenient tissues for confirma-
308
tion of the underlying enzyme deficiency in inborn errors of metabolism. Most of the enzymes found in skin fibroblasts are also expressed in cells cultured from amniotic fluid and chorionic villous tissue. Cultured cells and uncultured chorionic villi are suitable for prenatal diagnosis of inherited metabolic disorders, particularly those in amino acid metabolism. Abnormal amino acid patterns in amniotic fluid have only been found in argininosuccinate lyase deficiency (argininosuccinic acidemia) and argininosuccinate synthetase deficiency (citrullinemia). On the other hand, analysis of metabolites in amniotic fluid by the isotope dilution assay is the preferred technique for the prenatal diagnosis of many organic acidopathies. Sudden infant death (SID) is an enigma, and in most cases the underlying cause of death is unknown. Recently, fatty acid oxidation defects, particularly MCADD, have been incriminated as a fairly common cause of sudden death in infants over six months of age. The metabolic abnormalities that have been found in SID are listed in Table 6. Vitreous (aqueous) humor from these infants appears to be suitable for the detection of organic acid disorders (29). DNA DIAGNOSIS DNA diagnosis by restriction fragment length polymorphism (RFLP) and linkage analysis has been applied to prenatal diagnosis and to identification of the carrier state in individuals at risk for certain metabolic disorders. In families in which the gene mutation is known, direct DNA analysis for the mutation is the definitive diagnostic test. A good example is MCADD in which 90% of the patients have the same mutation, a transition of A to G985 (30). The polymerase chain reaction (PCR) is a rapid method to generate enough DNA for diagnostic studies (31) and can be used with as little as 5 to 10 ~LL of blood on filter paper (32). These techniques are particularly applicable to disorders in which the enzyme is not expressed in readily available tissue or in which diagnosis by metabolite pattern is unreliable. With a rapidly enlarging database on gene mutations in hereditary metabolic disorders, one can anticipate an increasing demand for gene diagnosis including newborn screening for these inborn errors of metabolism in the 1990s.
Acknowledgements This work was supported in part by the United States Public Health Grant NS 05096. The author is grateful to Ms. Roseann Mandell and Ms. Kathleen Kirsch for their assistance in the preparation of this manuscript.
References 1. Bickel H, Guthrie R, Hammersen G, eds. Neonatal screening for inborn errors of metabolism. Berlin: Springer-Verlag, 1980.
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HEREDITARY METABOLIC DISORDERS 2. Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Part 4 -- amino acids and Part 5 -- organic acids. In: The metabolic basis of inherited disease. 6th ed. Pp. 495-961. New York: McGraw-Hill, 1989. 3. Bremer HJ, Duran M, Kamerling JP, Przyrembel H, Wadman SK. Disturbances of amino acid metabolism: clinical chemistry and diagnosis. Baltimore: Urban & Schwarzenberg, Inc., 1981. 4. Fernandes J, Saudubray J-M, Tada K, eds. Part V -disorders of amino acids and organic acids. In: Inborn metabolic disease - diagnosis and treatment. Pp. 161-343. Berlin: Springer-Verlag, 1990. 5. Hommes FA, ed. Techniques in diagnostic human biochemical genetics - a laboratory manual. New York: Alan R. Liss, Inc., 1990. 6. Saudubray JM, Ogier H, Bonnefont JP, et al. Clinical approach to inherited metabolic diseases in the neonatal period: a 10-year survey. J Inherited Metab Dis 1989; 12: 25-41. 7. Blom W, Huijmans GM, van den Berg GB. A clinical biochemist's view of the investigation of suspected inherited metabolic disease. J. Inherited Metab Dis 1989; 12: 64-88. 8. Collins JE, Brenton DP. Pancreatitis and homocystinuria. J Inherited Metab Dis 1990; 13: 232-3. 9. Shih VE, Axel SM, Tewksbury JC, Watkins D, Cooper BA, Rosenblatt DS. Defective lysosomal disease of vitamin B12 (cblF): a hereditary cobalamin metabolic disorder associated with sudden death. A m J Med Genet 1989; 33: 555-63. 10. Stabler SP, Marcell PD, Podell ER, Allen RH. Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography -- mass spectrometry. Anal Biochem 1987; 162: 185-96. 11. Kang SS, Wong PWK, Cook HY, Norusis M, Messer JV. Protein-bound homocyst(e)ine -- a possible risk factor for coronary artery disease. J Clin Invest 1986; 77: 1482-6. 12. Chu RC, Hall CA. The total serum homocysteine as an indicator of vitamin B12 and folate status. A m J Clin Pathol 1988; 90: 446-9. 13. Hoffman G, Aramaki S, Blum-Hoffman E, Nyhan WL, Sweetman L. Quantitative analysis for organic acids in biological samples: batch isolation followed by gas chromatographic-mass spectrometric analysis. Clin Chem 1989; 35: 587-95. 14. Rinaldo P, O'Shea JJ, Welch RD, Tanaka K. Stable isotope dilution analysis of n-hexanoylglycine, 3-phenylpropionylglycine and suberylglycine in human urine using chemical ionization gas chromatography/mass spectrometry selected ion monitoring. Biomed Environ Mass Spectrom 1989; 18: 471-7. 15. Schmidt-Sommerfeld E, Wener D, Penn D. Carnitine plasma concentrations in 353 metabolically healthy children. Eur J Pediatr 1988; 147: 356-60. 16. Chalmers RA, Roe CR, Stacey TE, Hoppel CL. Urinary excretion of 1-carnitine and acylcarnitines by patients with disorders of organic acid metabolism:
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
evidence for secondary insufficiency of 1-carnitine. Pediatr Res 1984; 18: 1325-8. 17. Montgomery JA, Mamer OA. Measurement of urinary free and acylcarnitines: quantitative acylcarnitine profiling in normal humans and in several patients with metabolic errors. Anal Biochem 1989; 176: 85-95. 18. Wolf B, Grier RE, Allen RJ, et al. Phenotypic variation in biotinidase deficiency. J Pediatr 1983; 103: 233-7. 19. Heard GS, McVoy JRS, Wolf B. A screening method for biotinidase deficiency in newborns. Clin Chem 1984; 30: 125-7. 20. Wolf B, Heard GS. Screening for biotinidase deficiency in newborns: worldwide experience. Pediatrics 1990; 85: 512-7. 21. Kesner L, Aronson FL, Silverman M, Chart PC. Determination of orotic and dihydroorotic acids in biological fluids and tissues. Clin Chem 1975; 21: 353-5. 22. Brusilow SW, Hauser E. Simple method of measurement of orotic acid and orotidine in urine. J Chromatogr 1989; 493: 388-91. 23. Chalmers RA, Lawson AM. Organic acids in man. London: Chapman and Hall, 1982. 24. Rumsby G, Seakins JWT, Leonard JV. A simple screening test for medium-chain acylCoA dehydrogenase deficiency. Lancet 1986; 2:467 (Lett.). 25. Hauser ER, Finkelstein JE, Valle D, Brusilow SW. Allopurinol-induced orotidinuria -- a test for mutations at the ornithine carbamoyltransferase locus in women. N Engl J Med 1990; 322: 1641-5. 26. Pelet A, Rotig A, Bonaiti-Pellie C, et al. Carrier detection in a partially dominant X-linked disease: ornithine transcarbamylase deficiency. Hum Genet 1990; 84: 167-71. 27. Workshop Conference: Peroxisomes and Peroxisomal Disorders. J Clin Chem Clin Biochem 1989; 27: 287308. 28. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 1980; 102: 176-88. 29. Mills GA, Walker V, Ashton MR, Manning NJ, Pollitt RJ. Vitreous humour organic acids in medium chain acyl-CoA dehydrogenase deficiency. J Inherited Metab Dis 1990; 13: 239-40. 30. Yokota I, Indo Y, Coates PM, Tanaka K. Molecular basis of medium chain acyl-Coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation. J Clin Invest 1990; 86: 1000-1003. 31. Boehm CD. Use of polymerase chain reaction for diagnosis of inherited disorders. Clin Chem 1989; 3 5 : 1843-8. 32. McCabe ERB, H u a n g S-Z, Seltzer WK, Law ML. DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening. Hum Genet 1987; 75: 213-6.
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0009-9120/91$3.00 + .00 Copyright©1991The CanadianSocietyof Clinical Chemists.
Clin Biochem,
Detection of Hereditary Metabolic Disorders Involving Amino Acids and Organic Acids VIVIAN E. SHIH Amino Acid Disorder Laboratory, Massachusetts General Hospital, Boston, MA, USA, and Department of Neurology, Harvard Medical School, Boston, MA, USA Inherited disorders in the metabolism of amino acids and organic acids may cause neurological dysfunction and acute metabolic crises. For many of these disorders, early diagnosis and early treatment can greatly improve the outcome. A general description of the clinical manifestations and a discussion of selected techniques and approaches for the laboratory diagnosis are reviewed.
ratory diagnosis. Readers are referred to several excellent publications which include detailed descriptions of both the clinical and biochemical findings (2-4) as well as the laboratory techniques (5-7).
KEY WORDS: inborn errors of metabolism; amino acids; laboratory diagnosis.
Clinical m a n i f e s t a t i o n s
Introduction
n the past three decades, the clinical applicaIresulted tion of analytical biochemical techniques has in the discovery of many inborn errors of amino acid and organic acid metabolism. At least 150 disorders are now known and together their frequency is estimated to be in the order of I in 2,000 to 1 in 3,000 newborns (1). These metabolic disorders are of clinical importance because many cause severe clinical diseases. Many also respond well to treatment. Laboratory detection of inborn errors of amino and organic acid metabolism is now available at various medical centers and commercial laboratories. Screening for these disorders is carried out by various chromatographic techniques to detect the changes in metabolite pattern resulting from an enzyme block. Due to the rarity and complexity of some of these metabolic disorders, the referring clinician often relies upon the clinical biochemist to interpret the laboratory findings and advise on further diagnostic workup and management. It is now standard practice to include such interpretations when reporting results of metabolic screening. A general description of the clinical manifestations of the disorders of amino acid and organic acid metabolism will be followed by a discussion of selected techniques and approaches for their labo-
Correspondence: Vivian E. Shih, Massachusetts General Hospital, Amino Acid Laboratory, Bldg. 149, 13th Street, Boston, MA 02129, USA. M a n u s c r i p t received December 21, 1990; revised March 4, 1991; accepted April 24, 1991. CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
Disorders of amino acid and organic acid metabolism are, with a few exceptions, autosomal recessively inherited traits. The carrier parents are usually normal and, with each pregnancy, there is a 1-in-4 chance of an affected child. The clinical manifestations of these metabblic disorders vary considerably from neonatal death to mild neuropsychiatric symptoms in adulthood. However, the majority of patients come to medical attention in infancy or childhood. Many of these disorders affect the central nervous system, and the most common and nonspecific finding is developmental delay. Hypotonia, myoclonic seizures and choreoathetoid movements have been observed in a number of metabolic disorders, particularly those involving organic acids. Spastic paraparesis and awkward gait can be late complications of hyperammonemia. Infection often precipitates episodes of vomiting, ataxia and lethargy with progression to coma in patients with a number of amino acid and organic acid disorders. Such acute metabolic encephalopathy due to hyperammonemia or metabolic ketoacidosis is a life-threatening complication. Other clinical features suggesting the need for metabolic screening include dislocated optical lenses, unexplained hepatomegaly or cardiomyopathy, and vascular occlusion. Symptoms of acute pancreatitis in children have recently been seen in association with several metabolic disorders (methylmalonic acidemia, maple syrup urine disease, isovaleric acidemia, and homocystinuria) (8). Mild facial dysmorphism and anomalies such as cystic kidney have now been described in several organic disorders, including lactic acidosis, glutaric acidemia Type II, and cobalamin F mutant (2,9). Macrocephaly is a prominent sign in N-acetylaspartic aciduria associated with Canavan's disease. 301
SHIH TABLE 1
Simple General Urine Screening Tests Inspection for color and odor Dinitrophenylhydrazine (DNPH) and/or FeC13 test for a-keto acids Cyanide-nitroprusside test for disulfides Test for reducing substances Test for ketones Dipstick for sulfite
It is notable that not all amino acidurias cause clinical disease. Many of the following disorders have been detected in asymptomatic individuals by routine newborn screening and family survey: histidinemia, hyperprolinemia type 1, cystathioninemia, hyperlysinemia, Hartnup disorder, familial renal iminoglycinuria and dicarboxylic aminoaciduria. Early diagnosis is important as early treatment can prevent brain damage in many of these disorders. Long-term management includes nutritional therapy and/or megavitamin administration. Acute metabolic decompensation associated with hyperammonemia or metabolic ketoacidosis requires aggressive treatment including peritoneal dialysis or hemodialysis. Organ transplant is a promising new form of enzyme therapy.
Laboratory detection Specimen collection is a very important step in the detection of metabolic disorders. In acutely ill patients, the blood and urine specimens on admission are most appropriate for metabolic screening and should be saved from all patients whose diagnosis is unclear. Blood chemistry such as gases, pH, electrolytes, anion gap, glucose and ammonia provides clues to the nature of the metabolic disorder. These data as well as a brief clinical resume should be made available to the biochemist for interpretation of the screening results. Routine metabolic screening usually includes several simple preliminary tests and chromatographic analysis of amino acids and organic acids. Serum carnitine levels and biotinidase screening are useful ancillary tests. Other more specific tests depend on the clinical findings and initial test results. PRELIMINARY GENERAL SCREENING TESTS
All urine specimens for metabolic screening are subjected to a battery of simple general screening tests (Table 1). Inspection of urine for color and odor often gives important clues to a metabolic derangement. For example, characteristic odors are found in maple syrup urine disease (MSUD) and sweaty feet syndrome (isovaleric acidemia) which
302
are disorders of leucine metabolism, and fish odor syndrome which is due to the accumulation of trimethylamine. The dinitrophenylhydrazine (DNPH) and ferric chloride tests detect alpha-ketoacids in disorders such as phenylketonuria (PKU), MSUD, and tyrosinemia I. The cyanide nitroprusside test detects disulfides such as cystine, homocystine and 3-mercaptolactate-cysteine mixed disulfide. Medications containing sulfhydryl group such as captopril, penicillamine, dimercaptosuccinic acid, and N-acetylcysteine react with the nitroprusside reagent and interfere with this test. The presence of reducing substances and ketone bodies in urine can be detected by commercially available reagent tablets and test strips. Sulfite screening should be performed only on a fresh urine sample, and preferably in combination with thiosulfate determination, to detect molybdenum cofactor deficiency/ sulfite oxidase deficiency. A M I N O ACID ANALYSIS
Urine amino acid screening is performed in our laboratory using high voltage electrophoresis at pH 1.9 followed by paper chromatography in butanol-acetic acid-water (12:3:5) for two-dimensional separation (5). Thin-layer chromatography is an alternative method. Quantitative analysis is used by some laboratories for screening. Blood amino acids are quantified on an amino acid analyzer or a high-performance liquid chromatography (HPLC) system. The technique should be sensitive enough to measure amino acid concentrations as low as 3 ~moles/L. This limit is important because a low citrulline level is a diagnostic criterion of defects in urea synthesis. Free argininosuccinate in plasma or amniotic fluid can be difficult to detect and quantify since it often coelutes with another compound. Therefore, the sample should be boiled in acid before analysis to convert the free acid to its stable anhydrides. In normal individuals, circulating homocysteine, which has a sulphydryl group, is bound to serum proteins. In pathological conditions, the bound homocysteine is likely to be increased before the free disulphide form, i.e., homocystine, accumulates. Thus, measurement of total (free + bound) homocysteine rather than only free homocystine is a more sensitive diagnostic test for mild homocyst(e)inemia. Several methods have been developed for quantifying total homocysteine and cysteine, and all involve reduction and release of these protein-bound amino acids by a thiol reagent before analysis (10:12). ORGANIC ACID ANALYSIS
Gas chromatography using capillary column in combination with mass spectrometry (GC/MS) is
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
HEREDITARY METABOLIC DISORDERS
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Figure 1--Comparison of urine organic acid profiles in medium-chain acyl-CoA dehydrogenase deficiency (MCAD DEF., top) and consumption of dietary medium-chain triglycerides (bottom). the preferred technique for organic acid analysis. Table-top models with automated computerized analysis of the mass spectra are now available at affordable prices. Organic acids are isolated from blood and urine by solvent extraction, anion exchange column, or batch isolation using silicic acid before derivatization to the trimethylsilyl or methyl esters. Oxime formation is useful for the detection of ketoacids, aldehydes and ketones. Quantification of organic acids is notoriously inaccurate, largely due to inconsistent efficiency of extraction. Hoffmann et al. (13) recently described an improved method for sample preparation suitable for quantitative analysis. Measurements using selected ion monitoring and stable isotope dilution are sensitive and accurate techniques (14) which are applicable to prenatal diagnosis of organic acid disorders. Screening for volatile short chain fatty acids is not the test of choice for detection of the short chain fatty acid disorders such as isovaleric aci-
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
demia or propionic acidemia. The volatile fatty acids are often not increased during remission and the diagnosis can best be made by detecting their nonvolatile glycine conjugates (e.g., isovalerylglycine) and other characteristic metabolites (e.g., methylcitrate or 3-hydroxypropionate) which are present in blood and urine at all times. CARNITINE MEASUREMENT
Carnitine plays an important role in transporting fatty acids into and out of mitochondria where their oxidation takes place. Total serum carnitine consists of two fractions, free and esterified (5,15). A lower than normal ratio of the free-to-esterified serum carnitine signifies the presence of excessive organic acids. The total serum carnitine may be decreased in organic acidurias. Slightly reduced serum free carnitine is common in patients on valproate treatment or on a ketogenic diet. Analysis of acylcarnitines in the urine by fast atom born-
303
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Figure 2--Evolution of urine organic acid pattern during acute metabolic decompensation in propionic acidemia. Top, on admission; middle, after one day of treatment; bottom, after three days of treatment.
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CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
HEREDITARY METABOLIC DISORDERS TABLE 2 Differential Diagnosis of Urea Cycle Disorders Blood Plasma Diagnostic Urine Ammonia Citrulline Amino Acids Orotic Acid
Deficiency N-acetylglutamate synthetase Carbamylphosphate synthetase Ornithine transcarbamylase Argininosuccinate (ASA) synthetase ASA lyase Arginase
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impregnated on filter paper. The frequency of biotinidase deficiency, as detected by newborn screening, is approximately one in 60,000, half with profound deficiency (20). Since biotinidase deficiency can result in significant clinical disability, for which simple and effective t r e a t m e n t is available, screening for this disorder among children with unexplained multisystem problems is justified.
b e e n a useful t e c h n i q u e in the diagnosis of a n u m ber of organic acid disorders. BIOTINIDASE SCREENING
Biotin is a vitamin cofactor for four carboxylases. Biotinidase hydrolyzes biotin from biotinyl peptides and biocytin, and is responsible for the recycling of biotin. Deficient biotinidase activity results in biotin deficiency. Many clinical manifestations are associated with biotinidase deficiency including seizures, mental retardation, skin rashes, vision and hearing impairment, loss of hair, as well as an organic aciduria (18). Heard and colleagues (19) have developed a semiquantitative screening test for biotinidase activity which requires only a drop of serum or blood
OROTIC ACIDMEASUREMENT
U r i n e orotic acid e x c r e t i o n is a v a l u a b l e test in the differential diagnosis of h y p e r a m m o n e m i c syndromes. T h e r e are t h r e e m e t h o d s for its m e a s u r e ment. In both t h e colorimetric m e a s u r e m e n t described by K e s n e r et al. (21) and the q u a n t i t a tive GC/MS m e t h o d r e c e n t l y described by Hoffm a n n et al. (13), sample p r e p a r a t i o n is by liquid
TABLE 3 Differential Diagnosis of Homocystinuria Urine
Plasma Methionine Cystathionine synthase deficiency
Methylation Defects Folate disorders Cobalamin disorders* cblC, cblD, cblF cblE, cblG
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*Eight genetic cobalamin mutants have been described: cblA and cblB affect only methylmalonate metabolism; cblC, cblD, and cblF affect both homocysteine and methylmalonate metabolism; cblE and cblG affect only homocysteine metabolism.
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SHIH TABLE 4 Causes of Neonatal Hyperphenylalaninemia Classic phenylketonuria Pterin cofacter disorders Mild persistent hyperphenylalaninemia Transient hyperphenylalaninemia Hereditary tyrosinemias Neonatal transient tyrosinemia Maternal phenylketonuria
ornithine transcarbamylase deficiency. An HPLC method described by Brusilow and H a u s e r (22) has the advantage of separating orotic acid and orotidine on a n anion exchange column. The elution time of these compounds is easily altered by column conditions, and proper identification of the peaks is essential.
Interpretation and discussion NORMALVARIATIONAND EFFECTSOF DIET, MEDICATION AND TREATMENT
partition chromatography on silicic acid. This, however, does not extract orotidine which is an important metabolite to monitor in carrier testing for
For proper interpretation of screening results it is important to recognize the normal variation of
TABLE 5 Metabolic Disorders Detected Over a 24-Month Period in Author's Laboratory
By Amino Acid Analysis Ornithine transcarbamylase deficiency1 Nonketotic hyperglycinemia Ornithine ~minotransferase deficiency (gyrate atrophy) Argininosuccinate lyase deficiency Carbamylphosphate synthetase deficiency1 Argininosuccinate synthase deficiency Arginase deficiency HHH (hyperornithinemia, hyperammonemia, homocitrullinuria) syndrome Homocystinuria (cystathionine ~-synthase deficiency) Hyperprolinemia type I Mild hyperphenylalaninemia Vitamin B12 disorder (cblC) Cystinuria Total
1 1 1 1 29
By Organic Acid Analysis Medium-chain acyl-CoA dehydrogenase deficiency Methylmalonic acidemia Propionic acidemia Glutaric acidemia type I (glutaryl CoA dehydrogenase deficiency) Glutaric acidemia type II Biotinidase deficiency 2-Methylacetoacetyl CoA thiolase deficiency 4-Hydroxybutyric acidemia (succinic semialdehyde dehydrogenase deficiency) Fumarase deficiency Ethylmalonic acidemia N-acetylaspartic aciduria Short bowel syndrome 2 Ethylene glycol poisoning2 Total
1 1 1 1 1 19
1Suspected by amino acid and orotic acid analysis and confirmed by enzyme assay. 2Acquired abnormalities.
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HEREDITARY METABOLIC DISORDERS TABLE 6
Inherited Metabolic Disorders Detected in Sudden Infant Death (SID) Fatty acid oxidation defects Urea cycle disorders Maple syrup urine diseases Biotinidase deficiency Nonketotic hyperglycinemia Vitamin B12 disorder (cblF) Glycogenesis defects Systemic carnitine deficiency 3-Hydroxy-methylglutaric aciduria Pyruvate dehydrogenase deficiency
the urinary amino acid pattern on the basis of age, diet and medication. Infants less than six months of age normally excrete large amounts of proline, hydroxyproline, and glycine, but this pattern is abnormal when present in older individuals. Homocitrulline in the urine of infant is usually from infant formula and is only rarely due to a metabolic disorder such as the hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome or hyperlysinemia. Urinary glycine is quite variable and is also affected by diet (e.g., gelatin) and medication (e.g., valproate). Persistent hyperglycinuria suggests carrier status for familial renal iminoglycinuria and is rarely associated with other hereditary syndromes such as autosomal dominant glucoglycinuria and X-linked hypophosphatasia (2). Organic acidopathy should always be ruled out as the cause of glycine accumulation. If nonketotic hyperglycinemia is suspected, the diagnosis should be confirmed by a decreased plasma/ CSF glycine ratio. Chalmers and Lawson have compiled extensive data on the urine organic acid pattern in health and disease (23). Neonates excrete larger quantities of ethylmalonate, alpha-ketoglutarate, and intermediary compounds of the critic acid cycle than do older infants or children. Organic aciduria of dietary origin can interfere with the interpretation of organic acid analysis. As illustrated in Figure 1, the C6-Clo dicarboxylic acids are increased in the urine from a normal infant fed a formula containing medium-chain triglycerides (MCT), as well as in the urine from a patient with medium chain acyl CoA dehydrogenase deficiency (MCADD). The diagnostic metabolites for this fatty acid oxidation defect are the acylglycine (14) or the acylcarnitine (16,17) derivatives of the dicarboxylic acids. Phenylpropionate is a product of intestinal flora and, when absorbed, is metabolized by mediumchain acyl CoA dehydrogenase. In patients with MCADD, this compound and its glycine conjugate can be found in the urine. Monitoring phenylpropionylglycine excretion following oral administration of phenylpropionate has been used as an aid in diagnosing MCADD (14,24).
CLINICAL BIOCHEMISTRY, VOLUME 24, AUGUST 1991
Metabolites of valproate, a popular anticonvulsant drug, are frequently encountered in the urine and can mask an underlying organic acid disorder if organic acid screening is performed without the use of mass spectrometry. An example of how the timing of sample collection changes the urine organic and acid pattern is illustrated in a patient with propionic acidemia admitted in acute metabolic decompensation (Figure 2). The top panel, on admission, shows large quantities of ketones as well as 3-hydroxyvalerate and 3-hydroxypropionate, a pattern often seen during metabolic decompensation in propionic acidemia. After one day of intravenous fluid and electrolyte therapy, the ketones disappeared and methylcitrate and tiglylglycine were prominent (middle panel); after 3 days, only a few organic acids (lower panel) were detected. The diagnosis could have been difficult if this last sample were the only one analyzed. DIFFERENTIAL DIAGNOSIS
Hyperammonemia, hypoglycemia and ketonuria often accompany organic acid disorders, and their presence is valuable in the differential diagnosis of these disorders. The blood ammonia level in propionic acidemia and methylmalonic acidemia can be as high as that in urea cycle disorders. Mild elevation of blood ammonia together with hypoglycemia and low-ketone production is characteristic of defects in fatty acid oxidation. Recurrent severe ketosis suggests a ketolytic defect due to ketothiolase deficiencies. Lactic acidemia/aciduria is common in children. Among the many causes are impaired gluconeogenesis, defects in pyruvate metabolism, mitochondrial diseases, and any cause of hypoxia or hypoperfusion. In patients presenting with an acute encephalitic picture and/or hyperammonemia, urea cycle enzymopathies should always be considered. Ornithine transcarbamylase deficiency, in particular, is relatively common. The differential diagnosis of urea cycle disorders is shown in Table 2. Although hyperammonemia is a prominent sign in these disorders, the blood ammonia level can be normal during remission and high only after meals. The diagnosis of argininosuccinate synthetase deficiency, argininosuccinate lyase deficiency, and arginase deficiency can easily be made by finding their respective characteristic amino acid abnormalities. In contrast, a low plasma citrulline level may be the only clue to deficiencies of N-acetylglutamate synthetase, carbamylphosphate synthetase, and ornithine transcarbamylase. The two synthetase deficiencies can be positively identified only by enzyme measurement, whereas the combination of hyperammonemia, hypocitrullinemia, and orotic aciduria almost always indicates orni-
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SHIH
thine transcarbamylase deficiency. Female heterozygotes of this X-linked disorder may be normal, mildly affected, or severely affected, depending on the random inactivation of the X-chromosome (Lyonization). Asymptomatic individuals can be identified by the allopurinol test (25), the protein loading test (26), or DNA analysis (26). Homocystinuria is a manifestation of at least eight inborn errors of metabolism (Table 3). Cystathionine ~-synthase deficiency is the most frequently seen and is often associated with a high methionine level. In contrast, in methylation defects the methionine level is normal or low, but cystathionine and methylmalonate may be slightly increased. The total homocystine level is often increased in acquired vitamin Ble and folate deficiencies (12) and as a side effect of medications such as methotrexate, 6-azauridine, and isonicotinic acid hydrazide. Protein-bound homocysteine has also been considered a possible risk factor for arteriosclerotic disease (11). Pipecolic acid, formed in the metabolism of lysine, is primarily metabolized by peroxisomes, and its presence is an indication of a derangement of peroxisomal metabolism (27). Newborn screening for P K U is mandated by law in the United States. Hyperphenylalaninemia in the newborn period is not a single entity. As listed in Table 4, classic P K U is one of seven types of hyperphenylalaninemia. All neonates with hyperphenylalaninemia should have a pterin screening (28) for cofactor deficiency. Increased blood phenylalanine can be due to benign persistent mild hyperphenylalaninemia, may be secondary to neonatal tyrosinemia, or may be a transient finding. Infants of a P K U woman untreated during pregnancy will have increased blood phenylalanine during the first 24 h of life. DISORDER DETECTED
Our laboratory receives blood and urine specimens for metabolic screening from hospitals in the Boston area and several neighboring states. During the past 24 months, we have performed approximately 4,800 tests for the detection of disorders of amino acid and organic acid metabolism. These samples originated from a selected high-risk group, and multiple tests were frequently ordered on the same patient. The disorders detected are listed in Table 5. In addition to these, there were 6 cases of renal Fanconi syndrome, 1 case of trimethylaminuria (fish odor syndrome), 35 cases of lactic aciduria and at least 48 cases of ketonuria with unconfirmed underlying defects. P R E N A T A L A N D P O S T M O R T E M DIAGNOSIS
Blood cells and cultured skin fibroblasts and lymphoblasts are convenient tissues for confirma-
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tion of the underlying enzyme deficiency in inborn errors of metabolism. Most of the enzymes found in skin fibroblasts are also expressed in cells cultured from amniotic fluid and chorionic villous tissue. Cultured cells and uncultured chorionic villi are suitable for prenatal diagnosis of inherited metabolic disorders, particularly those in amino acid metabolism. Abnormal amino acid patterns in amniotic fluid have only been found in argininosuccinate lyase deficiency (argininosuccinic acidemia) and argininosuccinate synthetase deficiency (citrullinemia). On the other hand, analysis of metabolites in amniotic fluid by the isotope dilution assay is the preferred technique for the prenatal diagnosis of many organic acidopathies. Sudden infant death (SID) is an enigma, and in most cases the underlying cause of death is unknown. Recently, fatty acid oxidation defects, particularly MCADD, have been incriminated as a fairly common cause of sudden death in infants over six months of age. The metabolic abnormalities that have been found in SID are listed in Table 6. Vitreous (aqueous) humor from these infants appears to be suitable for the detection of organic acid disorders (29). DNA DIAGNOSIS DNA diagnosis by restriction fragment length polymorphism (RFLP) and linkage analysis has been applied to prenatal diagnosis and to identification of the carrier state in individuals at risk for certain metabolic disorders. In families in which the gene mutation is known, direct DNA analysis for the mutation is the definitive diagnostic test. A good example is MCADD in which 90% of the patients have the same mutation, a transition of A to G985 (30). The polymerase chain reaction (PCR) is a rapid method to generate enough DNA for diagnostic studies (31) and can be used with as little as 5 to 10 ~LL of blood on filter paper (32). These techniques are particularly applicable to disorders in which the enzyme is not expressed in readily available tissue or in which diagnosis by metabolite pattern is unreliable. With a rapidly enlarging database on gene mutations in hereditary metabolic disorders, one can anticipate an increasing demand for gene diagnosis including newborn screening for these inborn errors of metabolism in the 1990s.
Acknowledgements This work was supported in part by the United States Public Health Grant NS 05096. The author is grateful to Ms. Roseann Mandell and Ms. Kathleen Kirsch for their assistance in the preparation of this manuscript.
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HEREDITARY METABOLIC DISORDERS 2. Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Part 4 -- amino acids and Part 5 -- organic acids. In: The metabolic basis of inherited disease. 6th ed. Pp. 495-961. New York: McGraw-Hill, 1989. 3. Bremer HJ, Duran M, Kamerling JP, Przyrembel H, Wadman SK. Disturbances of amino acid metabolism: clinical chemistry and diagnosis. Baltimore: Urban & Schwarzenberg, Inc., 1981. 4. Fernandes J, Saudubray J-M, Tada K, eds. Part V -disorders of amino acids and organic acids. In: Inborn metabolic disease - diagnosis and treatment. Pp. 161-343. Berlin: Springer-Verlag, 1990. 5. Hommes FA, ed. Techniques in diagnostic human biochemical genetics - a laboratory manual. New York: Alan R. Liss, Inc., 1990. 6. Saudubray JM, Ogier H, Bonnefont JP, et al. Clinical approach to inherited metabolic diseases in the neonatal period: a 10-year survey. J Inherited Metab Dis 1989; 12: 25-41. 7. Blom W, Huijmans GM, van den Berg GB. A clinical biochemist's view of the investigation of suspected inherited metabolic disease. J. Inherited Metab Dis 1989; 12: 64-88. 8. Collins JE, Brenton DP. Pancreatitis and homocystinuria. J Inherited Metab Dis 1990; 13: 232-3. 9. Shih VE, Axel SM, Tewksbury JC, Watkins D, Cooper BA, Rosenblatt DS. Defective lysosomal disease of vitamin B12 (cblF): a hereditary cobalamin metabolic disorder associated with sudden death. A m J Med Genet 1989; 33: 555-63. 10. Stabler SP, Marcell PD, Podell ER, Allen RH. Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography -- mass spectrometry. Anal Biochem 1987; 162: 185-96. 11. Kang SS, Wong PWK, Cook HY, Norusis M, Messer JV. Protein-bound homocyst(e)ine -- a possible risk factor for coronary artery disease. J Clin Invest 1986; 77: 1482-6. 12. Chu RC, Hall CA. The total serum homocysteine as an indicator of vitamin B12 and folate status. A m J Clin Pathol 1988; 90: 446-9. 13. Hoffman G, Aramaki S, Blum-Hoffman E, Nyhan WL, Sweetman L. Quantitative analysis for organic acids in biological samples: batch isolation followed by gas chromatographic-mass spectrometric analysis. Clin Chem 1989; 35: 587-95. 14. Rinaldo P, O'Shea JJ, Welch RD, Tanaka K. Stable isotope dilution analysis of n-hexanoylglycine, 3-phenylpropionylglycine and suberylglycine in human urine using chemical ionization gas chromatography/mass spectrometry selected ion monitoring. Biomed Environ Mass Spectrom 1989; 18: 471-7. 15. Schmidt-Sommerfeld E, Wener D, Penn D. Carnitine plasma concentrations in 353 metabolically healthy children. Eur J Pediatr 1988; 147: 356-60. 16. Chalmers RA, Roe CR, Stacey TE, Hoppel CL. Urinary excretion of 1-carnitine and acylcarnitines by patients with disorders of organic acid metabolism:
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evidence for secondary insufficiency of 1-carnitine. Pediatr Res 1984; 18: 1325-8. 17. Montgomery JA, Mamer OA. Measurement of urinary free and acylcarnitines: quantitative acylcarnitine profiling in normal humans and in several patients with metabolic errors. Anal Biochem 1989; 176: 85-95. 18. Wolf B, Grier RE, Allen RJ, et al. Phenotypic variation in biotinidase deficiency. J Pediatr 1983; 103: 233-7. 19. Heard GS, McVoy JRS, Wolf B. A screening method for biotinidase deficiency in newborns. Clin Chem 1984; 30: 125-7. 20. Wolf B, Heard GS. Screening for biotinidase deficiency in newborns: worldwide experience. Pediatrics 1990; 85: 512-7. 21. Kesner L, Aronson FL, Silverman M, Chart PC. Determination of orotic and dihydroorotic acids in biological fluids and tissues. Clin Chem 1975; 21: 353-5. 22. Brusilow SW, Hauser E. Simple method of measurement of orotic acid and orotidine in urine. J Chromatogr 1989; 493: 388-91. 23. Chalmers RA, Lawson AM. Organic acids in man. London: Chapman and Hall, 1982. 24. Rumsby G, Seakins JWT, Leonard JV. A simple screening test for medium-chain acylCoA dehydrogenase deficiency. Lancet 1986; 2:467 (Lett.). 25. Hauser ER, Finkelstein JE, Valle D, Brusilow SW. Allopurinol-induced orotidinuria -- a test for mutations at the ornithine carbamoyltransferase locus in women. N Engl J Med 1990; 322: 1641-5. 26. Pelet A, Rotig A, Bonaiti-Pellie C, et al. Carrier detection in a partially dominant X-linked disease: ornithine transcarbamylase deficiency. Hum Genet 1990; 84: 167-71. 27. Workshop Conference: Peroxisomes and Peroxisomal Disorders. J Clin Chem Clin Biochem 1989; 27: 287308. 28. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 1980; 102: 176-88. 29. Mills GA, Walker V, Ashton MR, Manning NJ, Pollitt RJ. Vitreous humour organic acids in medium chain acyl-CoA dehydrogenase deficiency. J Inherited Metab Dis 1990; 13: 239-40. 30. Yokota I, Indo Y, Coates PM, Tanaka K. Molecular basis of medium chain acyl-Coenzyme A dehydrogenase deficiency. An A to G transition at position 985 that causes a lysine-304 to glutamate substitution in the mature protein is the single prevalent mutation. J Clin Invest 1990; 86: 1000-1003. 31. Boehm CD. Use of polymerase chain reaction for diagnosis of inherited disorders. Clin Chem 1989; 3 5 : 1843-8. 32. McCabe ERB, H u a n g S-Z, Seltzer WK, Law ML. DNA microextraction from dried blood spots on filter paper blotters: potential applications to newborn screening. Hum Genet 1987; 75: 213-6.
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