Nutritional Aspects of Inborn Errors of Metabolism

Michel Vidailhet

Department of Pediatrics 3, Hôpital d'Enfants, 54511 Vandoeuvre-lès-Nancy, France

GENERALITIES

Introduction

Inherited metabolic disorders in volve différent nutritional aspects. On the one hand, the disease itself can impair normal nutrition. During the end of the fetal growth period and the first two years of life, the human brain grows at an impres- sive rate. This brain growth spurt period (1) is associated with a very high rate of protein synthesis that makes the central nervous system vulnérable to any interfér ence with protein synthesis. Biochemical insuit at this critical period may hâve a permanent effect on brain function. The accumulation of one or more substrates above an enzymatic block can affect normal metabolism and nutrition. For example, in amino acid disorders the high tissue levels of accumulated amino acid may competitively inhibit the trans port of other amino acids sharing the same transport mechanism. Amino acids in excess may interfère with the activity of enzymes involved in the metabolism of other amino acids. Short-chain fatty acids, oxo-acids, and other organic acids in- teract with ureogenesis, gluconeogenesis, pyruvate metabolism, etc. In fructose in tolérance, the accumulation of fructose-1-phosphate causes the trapping of inorganic phosphate. The fall in cellular ATP and ADP inhibits several enzymatic activities and results in altérations of energy metabolism. On the other hand, the metabolic block can induce severe deficiencies in one or more metabolites normally produced by the pathway. In ail urea cycle disorders, except arginase deficiency, arginine becomes essential; without arginine supplementation, stunted growth and hyperammonemia occur despite protein restriction. Undemutrition may be the conséquence of gênerai metabolic disturbances. Chrome hypoglycemia, lactic acidosis, and secondary hypercorticism explain the poor growth observed in glycogenosis type I. Severe and progressive impairments of liver function and, to a lesser degree, of rénal tubular function play a major rôle in the development of malnutrition, which appears in galactosemia, fructose intolérance, or in hereditary tyrosinosis.

205 206 INBORN ERRORS OF METABOUSM

Even in lysosomal disorders, stunted growth can begin in the first few weeks of life, as in Wolman disease, probably because of the importance of the liver and small bowel lésions in this disease. In transport mechanism disorders, levels of spécifie nutrients may be decreased, with, as a conséquence, symptoms of deficiency, as in acrodermatitis enteropathica secondary to zinc malabsorption. On the other hand, surprisingly enough, cystinuria-lysinuria does not resuit in lysine or arginine deficiency, despite the increased rénal excrétion and the intesti nal malabsorption of thèse amino acids. Such an apparent contradiction is now well explained by the importance of direct intestinal absorption of di- and tripeptides.

Dietetic Treatment

Because of the multiplicity of inborn errors of metabolism, it is not possible to review ail their nutritional drawbacks. Some metabolic disorders are harmless variations of normal metabolism (e.g., essential fructosuria, cystathioninuria), whereas others cause severe illness or mental détérioration early in life. The latter are often treatable by means of spécial diets and their nutritional aspects appear most important for clinical practice. A block in galactose, fructose, or amino acid metabolism leads to accumulation of thèse substrates and their metabolites, which can be prevented by spécifie restricted diets. Such diets may hâve to be continued for many years, sometimes indefinitely, as in maple syrup urine disease (MSUD), urea cycle disorders, galactosemia, etc. The prolonged use of a synthetic or semisynthetic diet may hâve adverse effects if the absolute and relative amounts of the différent nutrients are not well provided. Thèse diets must provide: (a) adéquate calorie intake; (b) minimum requirements of essential amino acids and nitrogen; (c) vitamins, minerais, and trace éléments in sufficient amounts; (d) normal products of the metabolic pathway that can no longer be produced and that hâve essential functions (e.g., tyrosine in phenylketonuria (PKU), arginine in urea cycle disorders, cysteine in homocystinuria, free glucose in glucose-6-phosphatase deficiency). The biodisposability and intestinal absorption of several nutrients, such as vitamins or trace éléments, may be very différent between synthetic diets and normal foodstuffs. Apart from the main ef fects intended by the treatment, it is important to be concerned about ail nutritional problems that may resuit from the disease and its dietetic therapy. According to the type of metabolic disorder one can distinguish several kinds of dietetic manipulations.

Supplementation

This is the easiest to perform. An example is zinc supplementation for acroder matitis enteropathica. In Menkes disease parenteral supplementation with copper gives much less satisfactory results. A particular group of inherited metabolic diseases is the group of vitamin- INBORN ERRORS OF METABOUSM 207 dépendent disorders. Several amino acidemias and organic acidemias are completely or partially cured by large doses of vitamins that are the precursors of spé cifie cofactors involved in defective enzymatic reactions. Table 1 lists thèse diseases. According to the kind of enzymatic deficiency, a metabolic disorder, as methylmalonic acidemia, may be fully or partially responsive or unresponsive to B12 vitamin therapy. In every instance it is important to try such cofactor therapy, which is easier to apply than restricted diets.

Exclusion

Such diets can be proposed when the nutrient to be excluded is not an essential one. This is the case for galactose (galactosemia, galactokinase deficiency, glycogenosis type I) and for fructose (fructose intolérance, fructose-1, 6-diphosphatase deficiency, glycogenosis type I).

Restriction

In disorders involving an essential amino acid, the diet must supply the minimal requirement for this amino acid that is restricted relative to the total protein intake. Most of the natural protein is replaced by a protein substitute deprived of this amino acid (Tables 2 and 3).

TABLE 1. Amino acid and organic acid disorders that may be responsive to vitamin therapy

Disease Vitamin Dose

Homocystinuria (cystathionine synthetase deficiency) Cystathioninuria Pyridoxine 100-150 mg/day Hyperoxaluria Xanthurenic aciduria Methylmalonic acidemia (MMA) (mutase deficiency) B 0.25-1 mg/day Homocystinuria + MMA + hypomethioninemia 12 Homocystinuria + hypomethioninemia Folie acid 10-50 mg/day Maple syrup urine disease Lactic acidosis (pyruvate dehydrogenâte [PDH] Thiamin 10-20 mg/day deficiency)

Dicarboxylic aciduria Riboflavin 100 mg/day Type 2 glutaric aciduria Hartnup disease Niacin 50-200 mg/day Hyperphenylalaninemia with dihydropterin synthetase BH„* 3-5 mg/kg deficiency Tyrosinemia C 50-100 mg/day Multiple carboxylase deficiency (3-methylcrotonylglycine, Biotin 10 mg/day propionic, lactic acids)

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TABLE 3. Low amino acid and galactose-free substitutes

Disorder Substrate lowered Name of Product

Cystinosis cystine (0.5 g/100 g) Albumaid X Cysf Maple syrup urine disease Branched-chain amino M.S.U.D. Aid· acids Leucidonfc M.S.U.D. 1 and 2e Homocystinuria (classical Methionine Albumaid X Meth* form) Methionaid" Hom 1 and 2e Tyrosinosis Phenylalanine and tyrosine Albumaid X Tyr* Tyrosinaid* Tyrosidon" Tyr 1 and 2e Histidinemia Histidine Histinaid* Histidon" Hist 1 and 2e Hyperlysinemia Lysine Lys 1 and 2e Urea cycle disorders Nonessential amino acids Essential Amino Acid Mixture* U.C.D. 1 and 2e Nitrogen Ketosteril" Propionic and Isoleucine, methionine, OS 1 and 2e Methylmalonic acidemias threonine, valine Galactosemia Galactose Galactomin 19* Nutramigen" Pregestimil· Isomil' Prosobee" Vegelact9

•Scientific Hospital Supplies (England) •"Nutricia (The Netherlands) cMilupa (Fédéral Republic of Germany) ''Fresenius (Fédéral Republic of Germany) "Mead-Johnson (United States) 'Ross (United States) «Gallia (France) For PKU, see Table 2.

A major danger of such diets is the occurrence of nutritional deficiencies, particularly a deficiency in the restricted amino acid. Phenylalanine deficiency owing to overtreatment of PKU has been well described. The baby fails to gain weight; de- velops a severe, red, cutaneous rash starting in the napkin région; vomits; becomes anorexie and léthargie; and may develop alopecia, edema, and fréquent infections. Death or définitive mental retardation may ensue. Deficiency of other essential amino acids has the same gênerai effect. In several diseases such as MSUD, propionic acidemia (PA), B12 unresponsive methylmalonic acidemia (MMA), and infantile tyrosinosis, the borderline between deficiency and excess is narrow, and monitoring is more difficult than in PKU. Low protein diets, in which total protein is restricted and replaced by nonprotein calories, are necessary in several diseases such as urea cycle disorders. They give 210 INBORN ERRORS OF METABOUSM way to protein lack and sometimes necessitate the use of an essential amino acid mixture or of keto- and hydroxy-analogues of essential amino acids.

Emergency Conditions

Some of the diseases manifest themselves in severe acute forms, particularly in the neonatal period. This is the case for several amino acid disorders like MSUD, urea cycle disorders, MMA, PA, and isovaleric acidemia (IVA). Certain clinical findings are suggestive of a hereditary metabolic disease that frequently mimics neonatal sepsis. An important négative finding is the absence of fetal or périnatal distress that might explain the observed abnormalities. Metabolic diseases are characterized by a silent interval of variable duration (5-6 days in MSUD; 2-4 days in PA, MMA, and IVA; 2-6 hr in severe pyruvate carboxylase deficiency). It is in the second stage that symptoms occur, at a time when placental élimination no longer plays its rôle. Several features are characteristic: Thèse include neurological manifestations such as altérations in consciousness (lethargy, altemating hyperexcitability, and somnolence), hypertonia, convulsions, and feeding prob lème. Other suggestive findings include vomiting, abnormal breath or urine odor, a hemorrhagic syndrome, and jaundice. A number of simple laboratory tests can be diagnostically helpful in newborns thought to hâve a hereditary metabolic disorder. Blood déterminations should include true glucose, electrolytes, acid-base equilibrium, ketonemia, clotting factors, calcium, transaminases, and amino acid chromatography. Several simple urinary tests are helpful, including déterminations of ketones (Acetest), reducing sugars (Clinitest), glucose (Clinistix), and alpha- keto-acids (2-4 DNPH test). Depending on the suspected etiology, more special- ized testing can be undertaken such as an ion exchange chromatography of amino acids and gas chromatography/mass spectrometry of urinary organic acids. The eti ology of thèse acute conditions is summarized in Table 4. In such acute conditions resulting from amino acidemias, organic acidemias, or hyperammonemias, temporary removal of protein from the diet and high energy intake coming from carbohydrates help to limit accumulation of toxic metabolites. Peritoneal dialysis and exchange transfusions are frequently necessary to remove toxic metabolites and/or to correct hyperammonemia or severe metabolic acidosis.

AMINO ACID DISORDERS

PKU

Définition and Generalities

PKU can no longer be defined as a phenylpyruvic oligophrenia, owing to the success of dietary measures in preventing the appearance of mental deficiency in this disease. PKU is a hereditary metabolic disorder transmitted as an autosomal récessive trait, and arises from the permanent inactivity of hepatic phenylalanine INBORN ERRORS OF METABOUSM 211

TABLE 4. Possible hereditary metabolic disorders causing neonatal neurological distress

With acidosis With ketosis {contd.) MSUD (mild acidosis) Methylmalonic acidemia Methylmalonic acidemia Propionic acidemia Propionic acidemia Isovaleric acidemia Isovaleric acidemia β-keto-thiolase deficiency β-keto-thiolase deficiency Pyruvate carboxylase deficiency Pyruvate carboxylase deficiency Multiple carboxylase deficiency Multiple carboxylase deficiency CoA transferase deficiency Type 2 glutaric aciduria With hyperammonemia HMG CoA lyase deficiency Urea cycle enzyme disorders Nonketotic C -C dicarboxylic aciduria 6 10 CPS I deficiency Congénital hyperlactacidemias OCT deficiency With hypoglycemia Arginosuccinate synthetase deficiency Methylmalonic acidemia Arginosuccinase deficiency Propionic acidemia Arginase deficiency Pyruvate carboxylase deficiency Organic acidemias Multiple carboxylase deficiency Methylmalonic acidemia Types 1 and 2 glutaric aciduria Propionic acidemia HMG CoA lyase deficiency Isovaleric acidemia (+.) Nonketotic C6-C10 dicarboxylic aciduria β-keto-thiolase deficiency With alkalosis Pyruvate carboxylase deficiency Urea cycle enzyme disorders Multiple carboxylase deficiency CPS I deficiency Type 2 glutaric aciduria OCT deficiency HMG CoA lyase deficiency Hyperornithinemia with homocitrullinemia Arginosuccinate synthetase deficiency Arginosuccinase deficiency Congénital lysine intolérance Arginase deficiency Isolated neurological distress With ketosis Glycine encephalopathy MSUD D-glyceric I acidemia

HMG, hydroxymethylglutaric; CPS, carbamyl-phosphate synthetase; OCT, ornithine-carbamyl transferase; MSUD, maple syrup urine disease.

hydroxylase. It is characterized by a phenylalaninemia exceeding 25 mg/100 ml (1,500 μιηοΐ/ΐ), normal tyrosinemia, and urinary excrétion of phenylpyruvic and orthohydroxyphenylacetic acids (PPA and Ο OH PA) when normal protein dietary conditions exist (3 g/kg/day in the neonatal period). It was Fôlling in 1934 (2) who discovered PKU, Jervis in 1947 (3) who demonstrated the deficiency in phenylalanine's hepatic hydroxylation, and Mitoma et al. (4) and Kaufman (5,6) who stud- ied the enzyme System responsible for its hydroxylation (Fig. 1). This System in volves an enzyme complex associating phenylalanine hydroxylase, dihydropteridine reductase (DHPR), and the cofactors NADH and tetrahydrobiopterin (5,6). In true PKU, phenylalanine hydroxylase itself is inactive, demonstrating less than 1% of its normal activity. Two important advances were made in the treatment of this disorder when Bickel et al. in 1953 (7) demonstrated the efficacy of a low phenylalanine diet, and when Guthrie and Susi in 1961 (8), proposed a simple neonatal screening procédure that was accurate and economical. In France, PKU occurs once in approximately 15,000 births; it is approximately the same in the United States (9). Besides classical PKU, one can observe atypical 212 1NB0RN ERRORS OF METABOUSM

PROTEINS

• ./>

PHENYLPYRUVIC ACID PARA HYDROXYPHENYLPYRUVIC ACID

HOMOGENTISICACID

ORTHO-HYDROXYPHENIYL ACETIC ACID

1 PHENYLALANINEHYDROXYLASE 2 PHENYLALANINE TRANSAMINASE 3 TYROSINEAMINOTRANSFERASE 4 PARA HYDROXY PHENYLPYRUVATE OXIDASE

FIG. 1. Simplifiée! schéma of phenylalanine metabolism.

PKU and persistent moderate hyperphenylalaninemias in which the enzymatic defect is less severe. Giittler has published a comprehensive study of the genetic hypothèses explaining the three principal phenotypes in phenylalanine hydroxylase deficiency (10).

Clinical Présentation and Diagnosis

The clinical présentation of nontreated PKU has been well defined. The infant seems completely normal during the neonatal period. In some cases, digestive problems and vomiting are noted in the first weeks. A delay in psychomotor development and even régressions become évident after several months; convulsive épisodes are noted in 25% of the cases. Other findings include a depigmentation of hair and irises, abnormal urine odor described as musty, and fréquent eczematous cutaneous lésions. In 96% to 98% of the cases, mental deficiency is severe with an IQ below 50. When treated early and properly followed, PKU has an excellent prognosis, and mental deficiency is avoidable. Long-term statistical studies comparing affected INBORN ERRORS OF METABOUSM 213 children and their nonaffected siblings nonetheless demonstrate a slightly lower IQ in PKU infants, even when treated early (11). In the early school years, deficiencies in language (12), fine motricity, and temporospatial organization can handicap the child (13). The diagnosis of PKU is biological, consisting of the analysis of plasma phenylalanine and tyrosine levels, and the analysis of urinary excrétion of certain metabolites including PPA and Ο OH PA. Frequently, immaturity in phenylalanine's transamination reaction can delay the excrétion of PPA. Conversely, one can observe hyperphenylalaninemias caused by immaturity, or véritable transitory PKU, characterized by a phenylalaninemia exceeding 25 mg/100 ml, normal tyrosinemia, and excrétion of PPA and Ο OH PA (Table 5). Ail of thèse disorders normalize with time despite the return to a normal protein diet. The possibility of immaturity and enzymatic variants (14) nécessitâtes systematic vérification of the diagnosis of true PKU at about 3 months of âge. An oral phenylalanine loading test (100 mg/kg) can distinguish between true PKU, atypical PKU, and transitory hyperphenylalaninemia. It is also necessary to systematically eliminate the possibility of hyperphenylalaninemia owing to tetrahydrobiopterin deficiency whether it results from a deficiency in DHPR or in biopterin synthesis.

Treatment

The treatment of PKU is based on dietary restriction of phenylalanine. How- ever, since phenylalanine is an essential amino acid, the residual intake must re main sufficient (approximately 200-300 mg/24 hr). Phenylalanine tolérance varies according to the infant and the severity of the enzyme deficiency. Plasma phenyl alanine levels are customarily maintained slightly above normal (2-6 mg/100 ml) in order to avoid the risks associated with deficiency. Thèse risks include anémia, growth stagnation, anorexia, cutaneous lésions, lethargy, and mental deficiency, and can be as detrimental as those caused by excess intake. The level at which phenylalaninemia should be maintained remains a subject of discussion. This type of restricted diet can be achieved using protein hydrolysates low in phenylalanine or mixtures of pure amino acids lacking phenylalanine (Table 2). Thèse products should always be administered along with natural protein sources (small quantities of milk, for example) to assure a minimal intake of phenylalanine. The follow-up of treated PKU infants includes periodic clinical and biological control: growth and weight gain, psychomotor development, and phenylalanine levels. The diet should be adapted to âge and laboratory findings (introduction of green vegetables, fruits, and spécial low protein foods: flour, noodles, bread, low protein cookies, etc.). The dietitian, psychologist, and biologist should collaborate with the pedia- trician in treating thèse children. Every patient receiving treatment for PKU must receive vitamin suppléments and the diet of each patient should be considered most carefully from this point of view. Some low phenylalanine products do not Φ m m Ο T3 Φ ω £•=0 + co + § + c •û •ΰ iï TJ < S < g

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c Φ φ CO ο co if ô co "2 f i ** It a. ce co li E Ε _ 3 CO Ν ι li 11 luο i δ c S ζ il ο φ CO 1 2 0U8 | É8 c c ι Φ >. s. φ φ ·1 φ £ co co Φ S: Si»5lK» ρ efi c PI ο ^ iop t £ ce tsïfsl •S ο S 2-Ë ω φ b 70« ο Q. CO y 3 g « Φ 2 S ctiv e ni nthesi s co -ο ο Jiφl c II CO >, 3 .2 CO Ό c Φ >* Ιο ο < -e "° Ω Ω ïtfïi ! 1 1« b H I- INBORN ERRORS OF METABOUSM 215 contain added vitamins, and it is therefore essential to give the complète supplé ment each day. The absorption of some minerai éléments, especially calcium and zinc, may be compromised by the substitution of vegetable proteins for méat and the exclusion of milk and dairy products from the diet. Concern over trace élément deficiency was confirmed by the work of Alexander et al. (15) who showed a deficiency of iron, copper, zinc, and manganèse in PKU children receiving a low phenylalanine diet. A new formulation for trace élément supplementation with an increased dosage of iron, copper, and zinc was suggested. Despite a daily supple mentation with 8 g of a minerai mixture, Taylor et al. (16) still observed signs of a relative zinc deficiency in hair and plasma of PKU patients that might resuit from a compétitive inhibition of absorption by copper or other metals supplemented in the diet. Except for diet restriction, the lives of thèse children should be as normal as possible (family life, peer contact, schooling, etc.). The majority of authorities advise discontinuation of dietary restrictions at the âge of 6 or 7 years. Hère again, some uncertainties persist. A growing number of workers including Bickel (17), advocate the continuation of strict dietary restriction until the âge of 12. A problem that will become increasingly important in the years to corne is that of PKU mothers. It has conclusively been proven that children of nontreated PKU mothers suf- fer from intrauterine growth retardation, microcephaly, mental deficiency, and car- diac malformations. According to Lenke and Levy (18), risks are elevated when phenylalaninemia levels exceed 15 mg/100 ml. Restricted diets, initiated early in pregnancy, and maintaining phenylalaninemia below 8 mg/100 ml, might insure the birth of normal infants. Komrower et al. hâve recently outlined the practical difficulties of initiating and maintaining such diets in many cases, and hâve em- phasized the need for prospective studies in this poorly understood domain (19).

Hyperphenylalaninemias Caused by Tetrahydrobiopterin (BH4) Deficiency

In 1975, Kaufman et al. (20) first reported a case of PKU due to DHPR defi ciency in an infant suffering from severe mental deficiency despite dietary restric tions that were initiated early and correctly followed. Since BH4 acts as a cofactor in other hydroxylation Systems (tyrosine hydroxylase and tryptophan hydroxylase) necessary for the synthesis of neurotransmitters (DOPA, norepinephrine, seroto- nin) it was proposed that DOPA, Carbidopa and 5-OH-tryptophan be added to the phenylalanine restricted diet (27). Two kinds of BH4-deficiencies can be distinguished (22).

Hyperphenylalaninemias caused by DHPR deficiency The deficiency in DHPR can be demonstrated in a variety of tissues. Contrary to phenylalanine hydroxylase deficiency, it can also be demonstrated in cultured fibroblasts. 216 INBORN ERRORS OF METABOUSM

Hyperphenylalaninemias caused by dihydrobiopterin synthesis deficiency In 1977, another type of malignant hyperphenylalaninemia was described, characterized by tetraplegia, myoclonic crises, and death despite early dietary restric tion and even though phenylalanine hydroxylase and DHPR activities remained normal. Several case studies since then hâve demonstrated a deficiency in dihy drobiopterin synthesis. This deficiency can resuit from dihydrobiopterin synthetase or from guanosine-triphosphate (GTP) cyclohydrolase I deficiencies (23). Some patients with a dihydrobiopterin synthetase deficiency can be treated ef- fectively by BH4 monotherapy (5 mg/kg/day) whereas, in most cases, neurotrans- mitter precursor supplementation is necessary. Although hyperphenylalaninemias caused by disorders in biopterin metabolism are relatively rare (1 in 100 cases of true PKU), their négative évolution and successful treatment by DOPA and 5-OH- tryptophan justify routine screening for them in cases of neonatal hyperphenylala ninemia. Several methods hâve been proposed (24). The most promising tech niques, both précise and nonaggressive, appear to be assays of reduced and oxi- dized biopterins in the urine and the biological assay of BH4 in blood using a Guthrie card (25).

Disorders of the Urea Cycle

Since the description of argininosuccinic aciduria by Allan et al. in 1958 (26), numerous enzyme disorders affecting the différent stages of ureogenesis hâve been identified (22): mitochondrial carbamyl-phosphate synthetase (CPSI) deficiency; ornithine-carbamyl transferase (OCT) deficiency; argininosuccinate synthetase (ASS) deficiency responsible for citrullinemia; argininosuccinate lyase (ASL) de ficiency, responsible for argininosuccinic aciduria; arginase deficiency responsible for argininemia. Recently a deficiency in N-acetylglutamate synthetase has been discovered. This enzyme is necessary for the synthesis of N-acetylglutamate, which, in turn, activâtes CPSI (Fig. 2). The overall prevalence of urea cycle disor ders is estimated to be 1 in 3,000 live births. Ail of thèse urea cycle disorders, except for OCT deficiency, are transmitted in an autosomal récessive manner. OCT deficiency, the most fréquent of them, is transmitted as a dominant sex- linked trait. Urea cycle disorders are not the only metabolic disorders capable of giving severe hyperammonemia. Other hereditary metabolic disorders are frequently accompanied by hyperammonemia even in the complète absence of hepatic failure. This is the case in methylmalonic, propionic, isovaleric, and methylacetoacetic acidemias, as well as in pyruvate carboxylase deficiency (28). Différent mechanisms hâve been proposed to explain the abnormalities in ureogen esis and resulting hyperammonemia encountered in thèse diseases (29). Whatever the mechanism, when hyperammonemia is paradoxically associated with metabolic acidosis and ketosis (more often it is associated with alkalosis), investigations should be oriented toward the organic acidemias. Other disorders can also give INBORN ERRORS OF METABOUSM 217

Glutamic acid

î NH3 + C02

ADP

--,> CARBAMYL-PHOSPHATE

OROTICACID

Argininosuccinic acid

PYRIMIDINES

1. Carbamyl-phosphate synthetase I IC-PS-I) 2. Ornithine-carbamyl transferase (O.C.T. ) 3. Argininosuccinate synthetase 4 Argininosuccinate-lvase 5. Arginase

FIG. 2. Schematic représentation of the urea cycle. hyperammonemia (Table 6): (a) congénital protein intolérance with hyperlysinuria caused by abnormalities in the transport of basic amino acids (30), (b) hyperam monemia with hyperornithinemia and homocitrullinuria (31), (c) congénital lysine intolérance (32), (d) inadéquate portai circulation (porto-caval shunting), transient hyperammonemia of the prématuré (33). For each of the enzyme disorders affecting the urea cycle, clinical forms with varying degrees of severity hâve been reported. Each corresponds to one of the différent enzyme variants in which enzyme activity can be more or less diminished.

Neonatal Forms

The neonatal forms are the most severe and are often rapidly fatal. Defective enzymatic activity is less than 10% of normal in thèse patients, in contrast to children with intermittent forms who hâve greater than 10% of normal activity. After a Ω α Ζ ζ α. α.

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symptom-free interval from 1 to several days after birth, the infant begins showing anorexia, vomiting, progressive altération of consciousness from lethargy to coma, and convulsions. Laboratory investigations reveal considérable ammonemia at levels substantially higher than those observed in severe hepatic failure, often sur- passing 700 μΜ/l. At the same time glutaminemia is elevated, as well as glutamic acid, alanine, and lysine. Blood urea nitrogen may remain at normal levels despite defective urea synthesis. Occasionally one observes an increase in transaminases and a decrease in coagulation factors of hepatic origin. Alkalosis is présent more often than ketosis or metabolic acidosis. The EEG shows nonspecific diffuse altér ations. The physiological defect in thèse infants is an inability to synthesize and excrète waste nitrogen in the form of urea. This defect allows nitrogenous precursors of urea, glutamine, alanine, and ammonium to accumulate. Life-threatening épisodes of hyperammonemia can be treated with peritoneal or hemodialysis before irré versible damage occurs. In disorders other than arginase denciency, arginine, which is the only amino acid of the urea cycle to be incorporated into protein, becomes essential. Plasma arginine levels are low. A low protein diet given without arginine suppléments should increase the tendency toward arginine denciency, poor growth, and, in turn, reduced utilization of nitrogen, thereby tending to in crease hyperammonemia. There is also some évidence that arginine does stimulate activity of N-acetylglutamate synthetase, an enzyme responsible for production of N-acetylglutamate, which is the essential cofactor of CPSI. Moreover, as arginine is the main precursor of ornithine, required for uptake of carbamyl-phosphate, ar ginine supplementation allows urinary excrétion of both nitrogen atoms (coming from carbamyl-phosphate and aspartic acid) as argininosuccinic acid in ASL den ciency, and of one of them as citrulline in ASS denciency. Whatever the mechanisms involved, arginine suppléments added to a reduced protein intake benefit patients with urea cycle defects other than arginase den ciency. One should aim at plasma arginine levels of 60 to 100 μπιοΐ/ΐ. In severe forms of CPS, OCT, and ASS deficiencies, additional measures are necessary. One approach is to replace natural protein by a mixture of essential L-amino acids. Another theoretically more effective approach replaces natural protein by a mix ture of keto- or hydroxyanalogues of several essential amino acids (leucine, isoleucine, valine, phenylalanine, and methionine) given with a mixture of other essen tial amino acids (threonine, lysine, tryptophan, and histidine) and L-arginine. Thèse approaches allow réduction of nitrogen intake, stimulate nitrogen utilization for synthesis of nonessential amino acids, and of some of the essential ones, from their keto-analogues (34). However, high costs of keto-analogue mixtures argue against their long-term utilization. More promising are treatments with sodium benzoate and phenylacetate that stimulate urinary waste nitrogen excrétion in the form of hippuric acid (benzoic acid + glycine) and phenylacetylglutamine (phenyl- acetic acid + glutamine), greatly decreasing the level of hyperammonemia (35,36). In the liver mitochondria, the benzoate forms benzoyl-CoA which is excreted with a high clearance by the kidney. If glycine is low, benzoyl-CoA will accumulate 220 INBORN ERRORS OF METABOUSM and impair gluconeogenesis, lipid metabolism, and carbamyl-phosphate synthesis: therefore, benzoate should be used only if organic acidemias hâve been ruled out as causes of the hyperammonemia. The use of phenylacetate is still expérimental. Its advantage, in relation to benzoate, is the fact that 2 mol of nitrogen are fixed per mol of phenylacetate. However, its répulsive smell prevents its pérorai admin istration at the présent time. During hyperammonemic coma, an intravenous infusion of sodium benzoate (250 mg/kg in a 3% solution) and of arginine hydrochloride (800 mg/kg) is followed by constant infusion. Peritoneal dialysis or hemodialysis appears to be much more efficient than exchange transfusion for ammonia removal. To reduce the cat- abolic state and endogenous utilization of protein for energy production that increases NH3 production, it is urgent to cover calorie needs with IV administration of glucose (80 ml/kg/24 hr of 20% glucose with electrolytes) and lipid emulsion (3 g/kg/24 hr), the latter after organic acidemias hâve been ruled out. Enterai nutri tion is undertaken as soon as possible, by continuous naso-gastric infusion, to as sure a calorie intake exceeding 120 kcal/kg/24 hr. As soon as ammonemia has fallen below 200 μπιοΐ/ΐ, a mixture of essential amino acids (0.5-0.7 g/kg) with added milk protein (0.5-0.7 g/kg) should be given without delay. Long-term therapy differs according to the spécifie enzymatic defect. In argininosuccinic aciduria, a protein restricted diet (1.5 g/kg/day), supplemented with ar ginine (0.5-0.7 g/kg/day in 4 divided doses) allows for équilibration. In citrullinemia, the therapy associâtes essential amino acids (0.5-0.7 g/kg/day), limited natural protein (0.5-0.7 g/kg/day), arginine (0.5-0.7 g/kg/day), and so dium benzoate (0.25 g/kg/day). Carbohydrates and lipids are provided in order to maintain calorie intake above 100 kcal/24 hr. In CPS and OCT deficiencies, citrulline should be used instead of arginine, in equimolar amounts: 1 mol of nitrogen will be fixed for argininosuccinate synthesis and released as urea (37). Acute épisodes of hyperammonemia, sometimes resulting in death, can occur despite this treatment. The precipitating causes include intercurrent infections or excessive protein intake. Treatment of thèse épisodes requires intravenous arginine and benzoate therapy together with peritoneal dialysis or hemodialysis when required. Some other treatments hâve been proposed: N-carbamylglutamate, which is an analogue of N-acetylglutamate, is effective in the treatment of hyperammonemia due to N-acetylglutamate synthetase deficiency and might improve control of am monia levels in OCT, ASS, and ASL deficiencies and in the partial deficiency of CPS (37). In argininemia, secondary to arginase deficiency, a low arginine diet started in the neonatal period normalizes arginine levels and allows normal intellectual and neurological development. Unfortunately in the neonatal forms of other urea cycle diseases, the results are far from this successful. Neurological and de- velopmental progress has been variable. Many children hâve delayed intellectual development; thèse unfortunate outeomes seem correlated with the duration and the severity of the neonatal hyperammonemic coma. INBORN ERRORS OF METABOL1SM 221

Glycine Encephalopathy

Neonatologists are familiar with this occurrence because of the rapid onset of the disease and its severity. The désignation glycine encephalopathy is preferred over hyperglycinemia without ketosis, initially proposed to contrast this disorder with the hyperglycinemias with ketosis. The latter is synonymous with several organic acidemias such as propionic and methylmalonic acidemias. In glycine en cephalopathy, hyperglycinemia can be moderate or even absent even though gly cine levels in the CSF are increased from 10 to 30 times normal values. Glycine encephalopathy is caused by the inactivity of glycine synthetase, the enzyme necessary for converting glycine to serine. The disease's severity probably stems from glycine's inhibitory rôle on synaptic transmission. After a free interval of 2 to 3 days after birth, severe neurological symptoms appear: extrême hypotonia, lethargy, and irregular superficial breathing. Respiratory failure often requires ventilatory assistance. The metabolic abnormalities include hyperglycinuria, moderate hyperglycinemia ranging from 6 to 14 mg/100 ml, and extrême increases in CSF glycine levels ranging from 1 to 2 mg/100 ml (N, 0.06 ±0.01 mg/100 ml). A ma jor élément in the diagnosis is the EEG demonstrating a flat tracing interrupted pseudoperiodically by bursts of sharp wave activity. The prognosis is not favor able. Should the infant survive the initial period of respiratory failure, thanks to modem methods of ventilatory assistance, severe mental deficiency with microcephaly and convulsions are nonetheless unavoidable. EEG findings subsequently evolve toward hypsarythmia. Différent therapeutic approaches hâve been tried sep- arately or together: exchange transfusion or peritoneal dialysis during acute épi sodes; semisynthetic low glycine-serine diet; choline, folie acid, N5-formyltetrahy- drofolate; methyl-serine; administration of sodium benzoate or ursodesoxycholic acid to increase glycine élimination (38). Treatment with strychnine, a spécifie antagonist of the glycine System in the CNS proposed by Gitzelmann et al. (39) has given better results even though it does not prevent severe mental retardation (40). D-glyceric acidemia described by Brandt and co-workers (41) appears with a very similar clinical and biochemical picture.

Branched-Chain Amino Acid Disorders

A great number of inborn errors of branched-chain amino acid metabolism hâve been described, nearly at every step of their catabolism (Fig. 3). Several variants of thèse diseases may be vitamin responsive: MSUD, thiamin responsive (20 mg/day) (42); MM A, vitamin B,2 responsive; PA, biotin responsive (10 mg/day), belonging in fact to a multicarboxylase deficiency with accumulation of other organic acids (3 methylcrotonylglycine, pyruvic, lactic, and 3 hydroxy- isovaleric acids). Vitamin therapy must be systematically tested even if, in severe neonatal forms, vitamin dependency is rarely observed (43). As in urea cycle dis- 222 INBORN ERRORS OF METABOLISM

I ι KÊTOISOCAPflOIC ACID a-KETO 0 METHYLVALEfitC ACID a KETOISOVALEHI'ISOVALE C ACID ETHYL> *' ISOVALERYL-CoA a-METHYLBUTYRYL-Co-t A ISOBUTYRYL-CoITYRYL A * 'LBUTY 3 METHYLCROTONYL CoA : METHACRYLYL-CoI A * 3LYL-C iCRYLY β-METHYLGLUTACONYL-CoA ο METHYL β-HYDROXYBUTYRY: L CoA (S-HYDROXYISOBUTYRYL-CQA YDRO> I *' ISOBU β HYDROXY-p-METHYLGLUTARYL CoA a-METHYLACETOACETYL CoA 0 HYDROXYISOBUTYRIC ACID I I

ACETOACETICACID METHYLMALONIC SEMIALDEHYDE

1. HYPERVALINEMIA (VAL1NE TRANSAMINASEJ PROPION YL-CoA

2 MAPLE SYRUP UfilNE DISEASE IBRANCHED KETOACIDS DECARBOXYLASE) DMETHYLMALONY'LMALOh L CoA 3 ISOVALERIC ACIDEMIA (ISOVALERYL-CoA ÛEHYDROGENASE I

4 Jl METHYLCROTONYL GLYCINURIA (β-METHYLCROTONYL CoACARBOXYLASE L METHYLMAUONYL-Co'LMALON A 5. 3 METHYLGLUTACONIC ACIDURIA |3 MG-CoA HYDRATASE I

6. β-ΗΥΟΗΟΧΥ-0-METHYLGLUTARIC ACIDURIA (HMG-CoA t-YASE) SUCCINYL-CoA

7 METHYLACETOACETIC ACIDURIA IK DEPENDENT KETOTHIOLASEI

B. PROPIONIC ACIDURIA j PROPION YL-CoA CARBOXYLASE1

9 METHYLMALONIC ACIDURIA IMM-CoA RACEMASE)

10 METHYLMALONIC ACIDURIA IMM-CoA MUTASEI

FIG. 3. Hereditary diseases of branched-chain amino acid metabolism. orders, the emergency treatment of branched-chain amino acid disorders during the acute phase has two mean goals: removal of toxic metabolites and anabolism. The more effective methods for removal differ, according to the toxic organic acids or amino acids accumulated; peritoneal dialysis associated with exchange transfusions in MSUD and in PA, forced diuresis and exchange transfusions in MMA, glycine supplementation in IVA (43). Carnitine supplementation may also be important to remove and to eliminate toxic organic acids such as acetylcarni- tines in MMA, PA, IVA, 3 hydroxy-3-methylglutaric aciduria, and β keto-thiolase deficiency (44). The arrest of catabolism and the promotion of anabolism needs a high calorie intake. Continuous enterai nutrition by a naso-gastric tube must be begun as soon as possible with carbohydrates (glucose and glucose polymers), lipids, vitamins, minerais, and precursor free amino acid mixtures. Precursor amino acids may be introduced in limited amounts after lowering of toxic metabolites. The long-term outeome of patients with thèse disorders is still disappointing when they are vitamin unresponsive. Many children die secondarily during an acute cri- sis, and many of the survivors are mentally retarded. A good outeome is possible for patients with MSUD if diagnosis is made early and treatment begun (45).

MSUD

Symptoms appear when plasma levels of the branched amino acids and their corresponding alpha-keto-acids increase, particularly when leucine levels exceed INBORN ERRORS OF METABOUSM 223

10 to 12 mg/100 ml. Treatment consists of maintaining the branched amino acid concentrations at approximately normal levels by implementing diets low in leucine, isoleucine, and valine. Since thèse three essential amino acids are necessary for normal protein synthesis, a minimal quantity of leucine, isoleucine, and valine must be présent in the diet. It is advisable to maintain the plasma levels of thèse three amino acids between 2 and 5 mg/100 ml, slightly higher than the normal values. In the classical acute form leucine tolérance is situated between 200 and 600 mg/day. Ail alimentary proteins contain substantial quantities of branched amino acids. Therefore, dietary restriction nécessitâtes the utilization of semisynthetic diets in which protein requirements are essentially provided by amino acid mixtures totally lacking the three branched amino acids. Natural proteins must be strictly limited to quantities corresponding to the infant's tolérance to branched amino acids. Requirements in water, minerais, trace éléments, vitamins, fats, and carbohydrates must not be neglected. During periods of decompensation, high lev els of branched amino acids (especially leucine) should be rapidly lowered to less than 10 mg/100 ml. Peritoneal dialysis has been shown to be the most effective technique—even more effective than exchange transfusion. Dialysis sessions should not exceed 48 hr. When treatment is initiated early enough (within the first 10 days after birth) and when a satisfactory equilibrium is rapidly obtained and maintained, results tend to be excellent with normal intellectual development. The best results are obtained when there is a previous family history of MSUD. In thèse cases, the diagnosis is usually made much earlier, before appearance of clinical findings. Such was the case in three out of six personal observations. In the subacute and moderate forms of MSUD, branched amino acid tolérance is considerably higher than in the classical acute form. Leucine tolérance ranges from 900 to 1,200 mg/day. In certain patients, a simple low protein diet is an adé quate therapy. In the intermittent forms of MSUD normal protein diets can be maintained provided that protein intake is diminished or abolished during periods of infection or stress causing excessive catabolism.

DISORDERS OF CARBOHYDRATE METABOLISM

Thèse disorders include glycogenoses, glycogen synthetase deficiency, fructose and galactose intolérances, and enzyme disorders affecting gluconeogenesis such as type I glycogen storage disease and fructose-1, 6-diphosphatase and pyruvate carboxylase deficiencies. Furthermore certain enzyme defects in amino acid metabolism and fatty acid beta-oxidation (Table 4) can cause severe hypoglycemia.

Galactosemia

Galactosemia is a récessive autosomal disorder described by Goppert in 1917. Galactose-1-phosphate uridyl transferase deficiency was established by Kalckar et al. in 1956 (46). Its frequency is approximately 1 in 55,000 newborns. The affected infant usually appears normal at birth and symptoms develop within 3 or 224 INBORN ERRORS OF METABOUSM

4 days after institution of milk feeding. The early manifestations include jaundice, anorexia, vomiting, hypotonia, hepatomegaly, and susceptibility to infection. Escherichia coli septicemia is not uncommon in thèse infants. Ophthalmologic ex- amination demonstrates cataracts, and ail patients hâve some rénal tubular involve- ment characterized by proteinuria, generalized amino aciduria, and tubular acidosis. There is frequently hypermethioninemia, tyrosinemia, and tyrosyluria due to hepato-cellular insufficiency. The absence of succinylacetone in urine allows for the differential diagnosis with true tyrosinosis. Without treatment the hepato-cellu lar damage worsens; splenomegaly, ascites, edema, and hemorrhagic phenomena supervene and death occurs rapidly, subséquent to infection or hepatic failure. Absence of galactose-1-phosphate uridyl transferase activity in erythrocytes can be shown provided that the child has not been recently transfused. In such circumstances, the démonstration of a partial enzymatic deficiency in both parents' eryth rocytes may be helpful for diagnosis. Dietary management in galactosemia aims to suppress, as completely as possi ble, galactose from the diet, to avoid the accumulation of galactitol and galactose- 1-phosphate (Fig. 4). As cataracts are the only pathological manifestation in galactokinase deficiency—one step earlier in galactose metabolism—galactose-1-phos-

GALACTOSE

UTP

1 GALACTOKINASE

2. GALACTOSE - 1 • P04 - URIDYL TRANSFERASE 3. U.D.P GLUCOSE 4 EPIMERASE 4 and 5- U.D.P GLUCOSE and U.D.P. GALACTOSE PYROPHOSPHORYLASE (presumed lo be identicall

FIG. 4. Galactose metabolism. 1NBORN ERRORS OF METABOLISM 225 phate accumulation appears as the main toxic compound for the liver, kidney, and brain. Galactose-1-phosphate inhibits phosphoglucomutase, pyrophosphorylase, and possibly glycogen phosphorylase. Galactose-1-phosphate accumulation probably induces a trapping of phosphorus with dégradation of adenine nucleotides resulting in increased urea production and impaired energy metabolism. Total élimination of galactose from the diet appears to be without adverse effects, despite its présence in a number of important body compounds as galacto- sphingolipids. In fact, the donor molécule of galactose during biosynthesis of thèse molécules (UDP galactose) can be synthesized from UDP glucose. Elimination of galactose is accomplished primarily by avoidance of milk and dairy products, which can be replaced by lactose-free milk substitutes. Beetroots, peas, liver, brain, sweetbread, or any organ méats are excluded as well as com mercial food products that may contain lactose or galactose. The immédiate effect of treatment is dramatic. Early treatment prevents liver and kidney failure, brain damage, and cataracts. Over a long term, total exclusion of galactose from the diet provides satisfactory growth and health. However, de spite early and well-followed treatment, many galactosemic children achieve only a low normal intelligence and expérience speech defects or visual perception difficulties. Most children make less intellectual progress than, for example, well- treated phenylketonurics. Furthermore, in galactosemic girls, the secondary risk of ovarian dysfunction appears to be important (47). Prénatal accumulation of galac tose-1-phosphate and galactitol has been demonstrated in the galactosemic fétus after abortion (48); this fact could well explain thèse disappointing results. Ac- cording to Gitzelmann and Steinmann (47), biosynthesis of galactose-1-phosphate from UDP glucose via UDP glucose-4-epimerase and UDP glucose-pyrophosphorylase (Fig. 4) might constitute a pathway for self-intoxication, not only in utero, but also later on.

Fructose Intolérance

Hereditary fructose intolérance is an autosomal récessive disorder characterized by a primary deficiency of hepatic aldolase (aldolase B) (Fig. 5). Most symptoms are explained by the accumulation of fructose-1-phosphate in those cells that me- tabolize fructose, namely hepatocytes, enterocytes, and rénal tubular cells. Fruc tose itself has no toxicity, as demonstrated by fructokinase deficiency, a metabolic disorder characterized by essential fructosuria, without any clinical symptoms. Symptoms occur when fructose, saccharose, or sorbitol are introduced in the diet. The classical case history is that of an infant that has been nursed for 3 or 4 months and is perfectly well. When vegetables, orange juice, or saccharose are added to the diet, the child begins to vomit, to be drowsy and léthargie after meals, and fails to thrive. If the diagnosis is not quickly suspected, severe liver damage will occur, with hepatomegaly, hemorrhages, lethargy, and, sometimes, edema, ascites, and splenomegaly. Jaundice may or may not be présent. In older 226 INBORN ERRORS OF METABOUSM

children and adults the diagnosis may be easier; the patients refuse anything sweet as they hâve already experienced nausea, vomiting, sweating, and dizziness after eating sweet food. The diagnosis can easily be made by an intravenous fructose tolérance test (0.25 g/kg) that induces hypoglycemia and hypophosphatemia. Enzymatic deficiency may be demonstrated by liver biopsy or, more easily now, by small intestinal biopsy (49). Therapy is simple and consists of the total élimination of fructose and ail poten- tial sources of fructose from the diet. Before using any formula, any drug, or any parenteral fluid, one should check carefully and make sure that it does not contain fructose (lévulose), sorbitol, or saccharose. As ail fruits, fruit juices, and a great number of vegetables are excluded, ascorbic acid supplementation may be proposed, even though vitamin C deficiency has not been reported in such patients.

Gluconeogenesis Disorders

Fructose-1, 6-Diphosphatase Deficiency

This autosomal récessive disease involves a one-way enzyme of gluconeogene sis permitting the cleavage of fructose-1, 6-diphosphate to fructose-6-phosphate (Fig. 5). Affected children remain euglycemic for 12 to 16 hr after the last meal until glycogen stores are depleted, after which they become hypoglycémie. A main problem in thèse children is lactic acidosis. The liver lacks the physiologie capac- ity to take up lactate originating from muscle, as well as glycerol released from adipose tissue, and to convert them into glucose. Fasting, stress, muscular exer cise, and infections precipitate lactic acidosis and, secondarily, hypoglycemia.

GALACTOSE 1 PO, —^ GLUCOSE 1 P04 41 ' GLUCOSE β PO„

FRUCTOSE ^ FRUCTOSE 1 PHOSPHATE FRUCTOSE 6 PHOSPHATE

} FRUCTOSE 1 6 DIPHOSPATE

! FHUCTOKINASE 4 GÎUCOSE 6 PHOSPHATASE

J FRUCTOSE 1 PHOSPHATEALDOLASE 5 PTRUVATE CARBOXVLASE

3 FRuCTOSL 1 6 DIPHOSPHATASE 6 PHOSPHOENQLPVRUVATE CARBOX¥K!NASE

FIG. 5 Fructose metabolism and gluconeogenesis disorders. INBORN ERRORS OF METABOUSM 227

Thèse children do not tolerate (but without aversion for sweet-tasting foods) fruc tose, which précipitâtes hypoglycemia, by the same mechanism as in fructose in tolérance. An intravenous fructose tolérance test induces hypoglycemia and hypophosphatemia, somewhat less severe than in fructose-1-phosphate aldolase deficiency. Therapeutic management of the disease is much more difficult than in fructose intolérance. Besides fructose exclusion, the treatment consists of fréquent feedings, every 2 to 3 hr, and active treatment of infections that can lead to severe lactic acidosis and hypoglycemia. In such circumstances continuous glucose infu sions by naso-gastric tube or IV perfusion may be necessary.

Glycogenosis Type I

Glycogen storage disease type I (Von Gierke disease) occurs because of the con génital inactivity of the key hepatic enzyme glucose-6-phosphatase that is normally active in hepatocytes, rénal tubular cells, and enterocytes. Individuals with the dis ease are unable to release free glucose into the blood and become hypoglycémie 2.5 to 3 hr after a meal. They accumulate glycogen and triacylglycerol in their liver and are unable to convert fructose and galactose into glucose (Fig. 5). Growth retardation, hepatomegaly, bleeding tendency, hyperlactacidemia, hyper- uricemia, and hyperlipidemia are the other main features of the disease. Stimulation tests with glucagon, epinephrine, galactose, and fructose clearly indicate that glucose cannot be released from glucose-6-phosphate. Since 1959, several cases hâve been reported in which in vitro glucose-6-phosphatase activity of frozen liver was normal, despite typical clinical and biological features. In such cases, named pseudotype I glycogenosis, repeated bacterial infections hâve been reported related to neutropenia, due to arrest of bone marrow maturation, and to neutrophil dysfunction (50). In thèse pseudo-type I glycogenoses, the apparently normal glucose-6-phosphatase activity disappears when enzymatic activity is as- sayed on intact microsomes coming from fresh unfrozen liver. A defect in glucose- 6-phosphate translocase, an enzyme that transports glucose-6-phosphate inside the microsome, has been demonstrated by Lange et al. (51). More recently, Nordlie et al. (52) reported another enzymatic type of pseudotype I glycogenosis, which they proposed to name type le glycogenosis. Whatever the exact enzymatic type, la, b, or c, the primary aim of treatment is to maintain glucose levels as well as possible. Since 1974, numerous publications hâve demonstrated that continuous nocturnal intragastric feeding, accounting for one-third of the total calorie intake, combined with fréquent daytime feeding, has prevented hypoglycemia and improved both growth and metabolic abnormalities. However, elevated blood lactate and triglycéride levels are not always completely corrected. Particular attention must be paid in the morning at the end of intragas tric infusion, because of the particular risk of hypoglycemia at this time. Children must be fed within 30 min after the cessation of intragastric infusion. Fructose and galactose must be maintained as low as possible in the diet as they are unable to produce free glucose. Recently Smit et al. (53) hâve shown the advantage of slow 228 INBORN ERRORS OF METABOUSM release carbohydrate such as uncooked cornstarch (2 g/kg) diluted in water, which maintains glucose levels for 6.5 to 9 hr. Uncooked cornstarch may be an effective alternative regimen when continuous nocturnal intragastric infusion is refused or badly tolerated.

DISORDERS OF FATTY ACID METABOLISM

In the last several years, a number of hereditary metabolic disorders affecting beta-oxidation hâve been reported. They are characterized by vomiting, altérations in consciousness, hepatomegaly, metabolic acidosis, elevated free fatty acids (FFA), and severe hypoglycemia without ketosis. Fat accumulâtes in liver and muscle. Thèse disorders présent acute neonatal forms, fatal within several days, as in certain cases of type 2 glutaric aciduria and C6-d4 dicarboxylic aciduria, or intermittent forms in which clinical, biological, and histological findings resemble those found in Reye syndrome (54). The patient's basic problem is the inability to maintain sufficient energy metabolism in circumstances with glucose depletion. The defective fatty acid oxidation induces defective hepatic gluconeogenesis (55) that, associated with a vanished sparing effect from the ketone bodies (56), is responsible for severe hypoglycemia. Treatment of thèse children aims at preventing starvation and, during acute épi sodes, at restoring as swiftly as possible normal glycémie levels via intravenous infusion of glucose.

Type 2 Glutaric Aciduria

The disorder type 2 glutaric aciduria (57,58), also termed ethylmalonic aciduria (59), is caused by a deficiency in acyl-CoA dehydrogenase activity that simulta- neously affects the catabolism of organic acids derived from branched-chain amino acids (isovaleric, isobutyric, alpha-methylbutyric acids) and tryptophan (glutaric acid) as well as the catabolism of medium-chain fatty acids (C6-C10 dicarboxylic aciduria) and butyric acid. The manifestations of this organic acidemia are very similar to those observed in Jamaican vomiting sickness, caused by hypoglycin, a végétal toxin that inhibits the same acyl-CoA dehydrogenases (60). Type 2 glutaric aciduria présents either as a severe neonatal form with vomiting, coma, sweaty feet odor, hypoglycemia, hyperammonemia, and metabolic acidosis (57,58,61), or as an intermittent form evolving in épisodes of nonketotic hypoglycemia (62). In vestigations indicate that the dehydrogenase apoenzyme is intact and that the defects are probably localized on the common flavoproteins (57,63).

Nonketotic C6-Cu Dicarboxylic Aciduria

Nonketotic dicarboxylic aciduria is not a well-defined disease, but indicates di carboxylic aciduria that is seen in congénital defects related to the beta-oxidation of fatty acids. It stands in contrast to the ketotic dicarboxylic aciduria observed in INBORN ERRORS OF METABOUSM 229 diabètes or starvation where beta-oxidation is increased and accompanied by ke- tonuria and C6-C8 dicarboxylic aciduria resulting from omega-oxidation of FFA. This entity clinically manifests itself as épisodes of lethargy or coma with hypoglycemia. The first patient reported died on the first day of life with lactic acidosis and C6-C)4 dicarboxylic aciduria (64). Five other patients survived the neonatal period but subsequently suffered a number of Reye syndrome-like attacks (65-67). The enzymatic defect probably involves the medium-chain acyl-CoA dehydrogenase. In 1977, Tanaka et al. (68) reported an observation characterized by acute crises with hypoglycemia, hyperammonemia, adipic aciduria, and mild Cg-Cio dicarbox ylic aciduria related to a butyrl-CoA dehydrogenase deficiency.

Systemic Carnitine Deficiency

This disorder manifests itself in a similar manner, with épisodes of acute encephalopathy, vomiting, lethargy, coma, hepatic failure, metabolic acidosis, nonketotic hypoglycemia, hepatic steatosis, and lipid myopathy. In acute crises, findings resemble those in Reye syndrome. Muscular weakness and predominantly proximal amyotrophy can be présent. Thèse abnormalities are due to a severe car nitine deficiency. Another finding, although inconstant, is C6-Ci0 dicarboxylic aciduria (69-71). Uncertainty still remains as to the origin of the carnitine defi ciency (72). Whatever it may be, carnitine therapy may be bénéficiai.

Carnitine Palmitoyl Transferase Deficiency

In the forms generally observed in adulthood (73), this disorder is primarily characterized by muscular abnormalities including muscular pain and myoglobinuria after strenuous physical activity. In the more commonly cited adolescent or adult forms, carnitine palmitoyl transferase (CPT) deficiency appears only to affect skeletal muscle while leaving other metabolic sites intact. Recently however, Bougnères and co-workers (74) reported an infant suffering from hepatic CPT de ficiency responsible for fasting hypoglycemia and hypoketonemia. Mention should also be made of three cases reported by Hermier et al. (75) in which an encephalopathy was associated with CPT deficiency.

DISORDERS IN THE METABOLISM OF TRACE ELEMENTS

Our understanding of the metabolism and physiological rôle of trace éléments has been increasing rapidly. A number of pathological entities hâve been attributed to trace élément disorders essentially involving iron, iodine, zinc, copper, sélé nium, and chromium. The rôle of other trace éléments such as cadmium, vana dium, molybdenum, and manganèse has also recently received attention. Trace éléments intervene in various domains including hematopoiesis, immu- 230 INBORN ERRORS OF METABOUSM

nity, thyroid hormones, metalloenzymes, oxidoreduction, glycoregulation, etc. We shall discuss only two hereditary disorders in trace élément metabolism encountered in infancy: acrodermatitis enteropathica and Menkes disease.

Acrodermatitis Enteropathica

This rare autosomal disorder appears in the first months after birth and is fatal when untreated (76). It is characterized by chronic diarrhea, hypotrophy, alopecia, periorificial and distal erythemato-squamous dermatitis, and récurrent infections. Zinc levels are usually low in blood, erythrocytes, and hair follicles (77). Metallo enzymes such as alkaline phosphatase are decreased. Zinc malabsorption is probably due to abnormalities in a zinc binding factor of enterocytic origin. The reason for the therapeutic efficacy of human milk compared with the deleterious effect of cow's milk has not been elucidated and has stimulated research in the rôle of exog- enous binding factors. Oral treatment with zinc sulfate (100 mg/day in divided doses) is rapidly effec tive. This treatment should be continued indefinitely, especially in pregnant pa tients, since zinc deficiency can cause abortion and malformations.

Menkes Disease

Also termed kinky-hair or, more exactly, steely-hair disease, Menkes disease is a rare récessive X-linked disorder affecting copper metabolism (78,79). Soon after birth, patients présent with a convulsive encephalopathy, abnormal (pili torti) and depigmented (steely) hair, hypothermia, growth deficiency, characteristic facial dysmorphy, and bone lésions (osteoporosis and metaphyseal fractures). It is char acterized by many of the features of copper deficiency but apparently without neutropenia and anémia. Death usually occurs before 2 years of âge. The copper de ficiency is caused by its abnormal intestinal absorption associated with its accumulation in gut mucosa. A large proportion of the small amount of copper absorbed accumulâtes in the kidney, probably during reabsorption in the rénal tubules. Treatment is based on parenteral administration of copper, which raises plasma copper and ceruloplasmin to normal but does not correct other facts of the disease. It may be more effective if initiated soon after birth.

REFERENCES

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DISCUSSION

Dr. Schwartz: My concern is not related to acute diagnosis and management, which in most tertiary care centers is sophisticated, but rather to the long-term management of pa tients. In the United States our problème corne from our inability to adequately define nutrients; specifically, foods are not well labeled. In the management, for example, of galactosemia and hereditary fructose intolérance, it is almost impossible to instruct the family to 234 INBORN ERRORS OF METABOUSM buy foods that hâve a known fructose or galactose content; we certainly avoid ail foods containing additives, even if it is never put on the label, hot dogs for example. Even for the pure foods, it is very difficult to exactly define the fructose content of spécifie foods. This is an area that requires further thought. I hâve no idea what goes on in Europe, but if it is as complicated as it is in the United States, it must also be very difficult to adequately manage thèse patients. Dr. Vidailhet: We also hâve some problems. Certain products produced by the agro- alimentary industry sometimes also contain toxic metabolites. Another important aspect of the long-term management is the family's understanding of the disease mechanism itself. Dr. Schwartz: Hâve you considered doing duplicate diet analysis in spécifie diseases? When you hâve a child on a particular diet, you can feed the child an equal quantity of the same food as put into a container, as well as what is refused; both can be analyzed. This is a familiar technique used in metabolic balance studies. I hâve not heard of anyone using it on an outpatient basis, but, theoretically, it could be a means for obtaining a spécifie measure of what, for example, the total fructose is in the diet of a child that is presumably on a fructose-free diet. Dr. Vidailhet: No, we did not use that technique. Dr. Stern: I think what you are describing is another way of measuring how much did I offer him, but how much did he actually take? Dr. Schwartz: There is another area that is particularly important for fructose intolérance. That is how to treat a child when he gets an infection. Fortunately, in the United States we can call up virtually any drug company day or night and find out exactly what the ingrédi ents are, but it poses significant problems because many of the liquid préparations do hâve fructose or sucrose as a filler. Dr. Vidailhet: We do not hâve any problem in France, because the drug composition is always clearly indicated. Dr. Stern: I do not think Dr. Schwartz was talking about the drug composition but rather about the vehicle in which the oral préparation for the antibiotics for young infants is given. This may hâve a large amount of fructose or sucrose, and that is not usually indicated on the label. Dr. Goyens: We should be very cautious, when comparing Menkes disease (MD) and copper deficiency, not to assimilate them. MD is much more complex than a copper deficiency; the copper metabolism in those patients is totally abnormal. Evidence can therefore be found in the différent symptomatology of MD and copper deficiency, the inefficiency of parenteral copper supplementation in MD and, probably more important, the abnormal han- dling of copper by cultured fibroblasts of MD patients. In fact, thèse fibroblasts hâve con- sistently elevated copper concentration compared to normal fibroblasts (1). Secondly, you mentioned zinc, copper, iron, and manganèse deficiency in treated PKU patients. There is, however, also a sélénium story in treated PKU patients that illustrâtes very clearly the problems encountered with synthetic exclusion diets. Ingrid Lombeck (2) in West Germany recently documented sélénium deficiency in thèse patients; we had the same findings in Brussels. The deficiency is due to the very low sélénium content of syn thetic diets used in thèse patients. Récent work done in Finland (3) and elsewhere (4) showed that sélénium supplementation with organic sélénium, selenium-enriched yeast, is much more effective than classical supplementation with sodium selenite or selenomethio- nine, not only in terms of plasma levels but also in terms of selenium-dependent enzymatic activities. However, yeasts are very rich in phenylalanine. As a conséquence, we are still unable to efficiently supplément PKU patients with organic sélénium. INBORN ERRORS OF METABOUSM 235

Dr. Stem: Dr. Goyens, what effects are seen from low sélénium? Dr. Goyens: Thèse PKU patients hâve very low plasma levels of sélénium and very low erythrocytic glutathione-peroxidase activity. They did not présent with clinical symptoms, but in biochemical terms, there are important abnormalities. Dr. Stem: The glutathione-peroxidase, for which sélénium appears to be a co-enzyme, has been demonstrated in fact as being missing in some newborns also because of sélénium deficiency. This study was done by Nathan Rudolph in Brooklyn several years ago. Unfor- tunately, no one has paid too much attention to it but it may be a cause of red cell breakdown. Is there any évidence in some of thèse patients that they hâve abnormal hemolysis? Dr. Goyens: I do not know about PKU patients, but the same hypothesis has been put forward to explain the shortened half-life for red blood cells in kwashiorkor patients (5). Dr. Bakken: I just want to make a couple of comments regarding PKU disease. This disease was discovered by a Norwegian doctor, A. Fôlling, 50 years ago. It has been followed very closely in Norway. You asked how long the patients should follow the diets. A study in older patients from 20 to 30 years, in which the disease was not detected and who are mentally retarded, shows that the behavior of thèse patients is very much improved by the diet. A second comment—at the Institute of Nutrition at the University of Oslo, a spécial program for families with PKU children was established that provided them with ail the information about the content of amino acids in différent foods and made it easier for them to know what to give and what not give, especially in the âge bracket 12 to 18 years when one may be a little bit less strict as far as the diet is concerned. They hâve now also started a program for other diseases such as MSUD. Dr. Vidailhet: The treatment of MSUD disease is much more difficult in the teenage period; it is frequently very difficult to obtain strict adhérence to the diet, although adhér ence to it is very important in MSUD. I personally discontinue the treatment after 12 years, in PKU. Dr. Bakken: We do not do the same now, because we saw that some of thèse patients, when 13 or 15 years old, perform poorly at school. We carried out a lot of psychological tests and other investigations, and it is obvious that some of them are better when remaining on the diet. We pediatricians hâve neglected the teenagers. They make up a very important group, but it seems that they do not belong anywhere. Dr. Stern: In the United States and Canada, and to some extent in Britain, the adolescent population belongs to the pediatricians, and it is now fairly rigorous that children under 18, at least as a minimum, are pédiatrie patients and not internai medicine patients. Neverthe- less, the adolescent problem is enormous. It is difficult enough to get an adolescent who is allergie to chocolaté to stop eating chocolaté at a party, but we hâve ail kinds of difficulties, for example, with adolescent diabetics who do not want to take their insulin. When you impose a very restricted diet in a peer pressure situation, it becomes an enormous social undertaking, not just a phenomenon of prescribing the diet and asking to hâve it. I suspect that one of the reasons for discontinuing the diet at 12 is to avoid having trouble with ado lescents who did not want to follow the diets, but Dr. Bakken's information is very interest- ing. It is difficult to obtain évidence for the necessity of a long-term diet on the basis of improvements of patients who hâve not been treated up until that point. There we are, in fact, facing the same problem as when giving iodine supplementation in iodine déficient cretinism. On the other hand, there is no good évidence for the initial discontinuation of the diets at either 6 or 12 years. If PKU is serious for the brain in the newborn, I know of no évidence that would suggest that it does not remain serious when the brain is getting older. I always wondered about the rationale for the discontinuation of the diets. I suspect 236 INBORN ERRORS OF METABOUSM that what our Norwegian colleague is saying may very well turn out to be correct, that détérioration may be far more subtle in terms of what happens but that there is so far no good évidence for discontinuation of the diet at any âge. Dr. Padilla Munoz: I hâve been fortunate to follow three families with PKU disease that were diagnosed very early and were treated with low phenylalanine diets. Thèse infants presented with severe neurological damage, with convulsive disorders and mental retardation. I hâve been lucky to follow some of thèse patients for more than 15 years. At the âge of 7 or 10 years, some of thèse cases were completely off of the low phenylalanine diet and did fairly well. They showed minimal brain dysfunction with slight motor disorder and low memory. One of them is already 17 years of âge and présents with hypogonadism. Could it be that some PKU patients, if they hâve been treated in the first 5 or 10 years, may do better afterwards and that some metabolic adjustments occur enabling thèse patients to do fairly well without any spécifie diet? Dr. Vidailhet: When PKU patients are treated very early, they do not présent with mental disturbances and the prognosis is very good, contrary to what happens in galactosemia. When treatment is ineffective, we need to exclude tetrahydrobiopterin (BU,) deficiency, which requires another treatment. The behavior of untreated PKU children does not improve when they get older, and they remain severely mentally retarded. However, in atypical PKU, when the levels of phenylalanine are not so high, maybe between 15 to 20 mg per dl, and when the patient is on a normal protein diet, the intelligence quotient may be better. Dr. Frenk: It is tempting to point out that in advanced malnutrition, one can find sorts of caricatures of several of thèse genetically determined metabolic disorders. Advanced malnutrition, particularly of the kwashiorkor (KWK) type, may, metabolically speaking, mimic many of thèse diseases. For instance, one of the particular characteristics of KWK is a transient deficiency of phenylalanine hydroxylase leading to a transient deficiency of tyrosine, which thus becomes an essential amino acid until recovery takes place (6). Since thèse metabolic caricatures are unfortunately much more common than the genetically de termined diseases, they provide a golden opportunity for investigation of the many still un- known features of the latter. Another example is the acrodermatitis enteropathica-like appearance of dermal and mucosal lésions in many KWK patients (7). We do not know much about minerai transport mechanisms in advanced malnutrition. Other examples of réversible biochemical and clinical features, similar to those of some genetically determined diseases, may be found (8) in many severe nutritional disorders. Dr. Goyens: I cannot agrée entirely with Dr. Frenk's comment. It is true that some symptoms and biochemical abnormalities observed in severe protein energy malnutrition sometimes mimic very well those observed in some inborn errors of metabolism. However, we should be very cautious. Indeed the term protein energy malnutrition (PEM) seems to imply a primary problem with dietary protein and energy, but our knowledge of the patho- physiology of PEM in différent parts of the world is still far from conclusive. For example, as far as zinc is concerned, I can hardly imagine how Golden's observations in Jamaica, and ours in Central Africa (9), could help expiai η the pathophysiological mechanisms of acrodermatitis enteropathica; it is rather the contrary! Dr. Maggioni: From a practical point of view, could you give us some data about the frequency of thèse metabolic diseases that require dietetic treatment, at least in France? Dr. Vidailhet: The most frequently encountered is PKU; its frequency (1 in 16,000) is well known thanks to the systematic neonatal screening. Urea cycle disorders are unfre- quently seen, mostly OCT deficiency and citrullinemia. Contrary to our colleagues in the United States we very seldom see cases of arginino succinic aciduria. Homocystinuria is INBORN ERRORS OF METABOUSM 237 very rare in France. Galactosemia is diagnosed in 1 in 50,000 neonates, MSUD 1 in 300,000. Dr. Schwartz: I would like to comment on that last issue because I hâve been impressed over the last 10 to 15 years that at least in the diagnosis of the urea cycle disorders, which are often fatal, some progress has been made. This is because our junior housestaff hâve been tuned into considering thèse defects and are making the diagnosis on the first or sec ond day of life in infants who previously died and in whom we could not make the biochemical diagnosis. Dr. Vidailhet: I agrée with your comment concerning the apparent frequency of inborn errors of metabolism. The more we know about thèse diseases, the more fréquent they become, particularly in the neonatal period. One should think of metabolic disorder any time the clinical picture suggests sepsis, which can easily be confused with metabolic disorders. However, we need to be very careful, because sepsis may also occur in a child with an inborn error of metabolism. The diagnosis of metabolic diseases can also be retarded when the child has had a transfusion. In galactosemia, if the enzymatic assay is done after a trans fusion, it may be quite normal. Dr. Marini: I agrée that we are now more oriented toward thèse diagnoses, but there is still a major problem with the very small preterm babies. When we see a full-term baby mimicking a respiratory distress syndrome (RSD)—urea cycle disorders mimic RSD rather than sepsis because they start with hyperventilation—after a few minutes, when you get the answer from the laboratory that blood ammonia is very high, it is not difficult to hâve a diagnosis, but it is not so easy with preterm infants. We do not really know the incidence of the disease in the very low birth weight infant. Also confusing is that in cases of propionic acidemia, they often hâve a very high tendency to hemorrhage; very small babies with propionic acidemia could be diagnosed as having intraventricular hemorrhage. Dr. Vidailhet: We hâve to think about propionic acidemia when a metabolic acidosis cannot be corrected with usual quantities of bicarbonate. As far as prématuré babies are concerned, you are right: Hyperammonemia may be due to an inherited metabolic disorder, or may be temporary, especially in children who had difficulties at birth, such as asphyxia and so on. Dr. Schwartz: I am concerned about the recommendation to use sodium benzoate in the treatment of severe hyperammonemia. As far as I know, it is expérimental. It has significant risks associated with it, and I do not think it should be presented as a clear form of therapy. I would like to see it presented as a question that needs further analysis. Dr. Vidailhet: The emergency treatment with sodium benzoate, in cases of OCT deficiency, is very efficient. We use it. There may sometimes be a problem with unconjugated bilirubin. Dr. Stern: Sodium benzoate is a powerful displacer of bilirubin from albumin; in fact, in the laboratory, it is the most potent one known. Caffeine sodium benzoate, which used to be used for that purpose experimentally, gets its effect from the sodium benzoate and not from the caffeine. That could be a problem only in a newbom with high bilirubin. Be- yond the newborn period, I imagine you could use it without any difficulty. Regarding PKU pregnant girls, in our department we hâve been working with monkeys producing PKU and looking at monkey offspring. There is no question that they are badly retarded in terms of performance and so on. People who hâve dealt with pregnant PKU patients hâve, however, found a great deal of difficulty in controlling the diet to get an adéquate protein intake during pregnancy and yet avoiding the outcomes. Some of them are so discouraged that they simply suggest that thèse girls do not get pregnant at ail. Could you comment on this phenomenon? 238 1NBORN ERRORS OF METABOUSM

Dr. Vidailhet: It is a very important problem now that more and more of our patients become teenagers. I stop the diet in boys, but maintain it partly for girls. I maintain a small quantity of spécial dietetic products. We now use Maxamaid XP, which is better in terms of palatability and is better accepted. We are not so strict anymore about the blood levels of phenylalanine after the âge of 12, but in the girls we maintain part of the protein intake in the form of a spécial product that is totally deprived of phenylalanine. It is very difficult to say to a teenage girl that she will never hâve children. Another thing is that the diet of pregnant PKU patients needs to be adjusted and again strictly controlled before conception and not 2 or 3 weeks later. Dr. Stern: The question 10 years ago was considered to be quite important in neonatal units regarding transient élévation· of tyrosine in small prématuré infants, which could be reversed with vitamin C and with folie acid, and there were papers showing that if this was not done, the children would not develop mentally as well as others. It was clearly related to the amount of protein that was given in the diet. Would you comment about whether this is still a real problem? Dr. Vidailhet: High tyrosine levels were observed when the protein intake was much more important, 4 to 5 g/kg/day. Now that the protein intake is lower, between 2.5 and 3.4 we do not observe high tyrosine levels anymore. Dr. Stern: Dr. Scriver and I found that below 3 g of protein/kg/day you rarely got into trouble, but if you did, ail you had to do was to give about 50 mg of vitamin C, which in most infants would induce the enzyme System very quickly, even in very small prématurés. Dr. Râihâ: It turns out that transient hypertyrosinemia in preterm infants is not really a problem of vitamin C because this acts on the second enzyme in the tyrosine oxidation pathway, on parahydroxyphenylpyruvate oxidase. The activity of that enzyme is normal in preterm infants. The problem is with tyrosine aminotransferase, whose activity is not so much affected by vitamin C. I do not really know if we can prevent that by just giving vitamin C. Dr. Stern: Can you induce it with anything? Dr. Ràihâ: Yes, you can induce tyrosine aminotransferase by giving the substrate. It can also be induced by corticosteroids. Dr. Stern: Does folie acid work at that level? Dr. Ràihà: I do not know that it does. Tyrosine aminotransferase is induced in vitro by corticosteroids, by thyroxin, and by substrate. Dr. Vidailhet: When we observe high tyrosine levels in prématuré babies, we see at the same time elevated levels of parahydroxyphenylpyruvic, lactic, and acetic acids in urine. This is in favor of parahydroxyphenylpyruvate oxidase deficiency much more than tyrosine transaminase deficiency. Dr. Vidyasagar: Until recently it has been legislated, at least in many states, that the infants should hâve 24 hr of feeding before PKU screening, but recently in Illinois it has been recommended that 24 hr would be sufficient with or without feeding because endogenous phenylalanine will show up. What is the expérience of other people? Dr. Vidailhet: We wait until the third day of life before screening. The phenylalanine level can go up, due to endogenous catabolism, but it is préférable not to do the screening beforehand. Dr. Stern: In other words, you think that if you do not feed the baby, it could not go up in the first 24 hr but may take about 72 hr. That has also been our expérience. We used to think it could take 5 days before we would get any élévation from endogenous breakdown of protein. INBORN ERRORS OF METABOUSM 239

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