Abstract
There are numerous disorders of amino acid (AA) metabolism; although individually rare, they collectively are extremely important. First, they may result in devastating disturbances of neurological development, and, second, they provide insight into normal and abnormal brain development. Disorders of AA metabolism refer to those in which the major accumulating metabolite is an AA, with the enzymatic defect primarily involving the initial step in the metabolic pathway. These disorders of AA metabolism (e.g., nonketotic hyperglycinemia [NKH], urea cycle defects, and maple syrup urine disease [MSUD]) are extremely important in the immediate neonatal period because if not recognized promptly, the propensity for devastating neurological consequences is markedly increased. In general the major clinical features of NKH, urea cycle defects, and MSUD include stupor, seizures, hiccups, vomiting, and dystonia. Treatment for NKH includes sodium benzoate to lower glycine blood levels; benzodiazepines, which enhance GABA receptor inhibitory function; and excitatory amino acid antagonists such as dextromethorphan to control seizures. Outcome of the severe neonatal form is generally poor with notable gender differences. Thus males demonstrate better outcomes as compared with females. For urea cycle defects, early identification and prompt treatment may improve survival, although the impact on long-term outcome remains unclear. Treatment strategies include removal of ammonia, which is best accomplished with hemodialysis; stimulating alternative pathways of waste nitrogen excretion (sodium benzoate, phenylbutyrate, or glyceroltriphenylbutyrate); and providing a low-protein diet with abundant nonprotein calories and essential AA. The potential role of gene therapy and liver transplantation remains unclear. The outcome with MSUD depends on early intervention (preferably <5 days) and includes control of leucine levels, optimizing specific amino acids that compete with branch chain amino acids for entry into brain.
Keywords
Maple syrup urine disease, Nonketotic hyperglycinemia, Phenylketonuria, Ornithine transcarbamylase deficiency, Carbamyl phosphate deficiency
Since the late 1950s, numerous disorders of amino acid metabolism have been described, with major implications for the developing nervous system. Although each of the disorders is rare, collectively they are important for two major reasons. First, they represent causes of devastating disturbances of neurological development that are potentially treatable, and second, they provide insight into normal and abnormal brain metabolism. Disorders of amino acid metabolism are defined, in this context, as those in which the major accumulating metabolite is an amino acid and the enzymatic defect involves the initial step (or, in one case, the second step) in the metabolism of the amino acid. In this chapter, those disorders of amino acid metabolism of especial importance in the neonatal period (e.g., maple syrup urine disease, nonketotic hyperglycinemia, and the urea cycle defects) are discussed in detail. Because urea cycle defects are characterized particularly by hyperammonemia, they are discussed in the larger context of neonatal hyperammonemia.
Overview of Aminoacidopathies With Neonatal Neurological Manifestations
Disorders of amino acid metabolism associated with neurological manifestations in the first month of life are shown in Table 27.1 . Many other disorders manifest later in infancy and childhood, including variants of most of those conditions listed in the table. The major clinical features include altered level of consciousness, seizures, vomiting (and impaired feeding), and delayed neurological development. In the following sections, maple syrup urine disease, nonketotic hyperglycinemia, and hyperammonemia, including the urea cycle defects, are emphasized because these represent the most common disorders. The other disorders in Table 27.1 are very rare and are noted only briefly (see the section on miscellaneous amino acid disorders later). Pyridoxine dependency is discussed in Chapter 12 .
| DISORDER | MAJOR CLINICAL FEATURES | ENZYMATIC DEFECT |
|---|---|---|
| Urea cycle defects a | Vomiting, stupor, seizures | Carbamyl phosphate synthase, ornithine transcarbamylase, argininosuccinic acid synthetase, argininosuccinase |
| Maple syrup urine disease a | Stupor, seizures, dystonia, odor of maple syrup | Branched-chain ketoacid decarboxylase |
| Nonketotic hyperglycinemia a | Stupor, seizures, hiccups | Glycine decarboxylase |
| Hypervalinemia | Stupor, delayed development | Valine transaminase |
| Phenylketonuria | Vomiting, musty odor | Phenylalanine hydroxylase |
| Lysinuric protein intolerance | Vomiting, hypotonia | Transport of cationic amino acids (lysine, arginine, ornithine) |
| Pyridoxine dependency b | Seizures | Glutamic acid decarboxylase (pyridoxal phosphate action), decreased gamma-aminobutyric acid synthesis |
a Most common disorders and discussed in this chapter.
b See Chapter 12 .
Maple Syrup Urine Disease
Maple syrup urine disease, in its classic form, is a fulminating neonatal neurological disorder caused by a disturbance in the metabolism of the branched-chain essential amino acids, leucine, isoleucine, and valine. The disturbance involves the second step in the degradation of these compounds (i.e., oxidative decarboxylation).
Normal Metabolic Aspects
Transamination
The first two steps in the degradation of the branched-chain amino acids (BCAAs) are shown in Fig. 27.1 . The initial transamination is thought to occur via a single transaminase. The usual amino acceptor for the transamination is alpha-ketoglutarate, which is converted to glutamate.
Oxidative Decarboxylation
The transaminations result in the formation of the three branched-chain ketoacids (BCKAs), which then undergo oxidative decarboxylation via a dehydrogenase complex to the corresponding short-chain fatty acids (see Fig. 27.1 ). Oxidative decarboxylation of the three alpha-ketoacids is particularly active in liver, kidney, heart, and brain. This reaction is a multistep sequence that requires thiamine pyrophosphate and lipoic acid. The former is of clinical importance because of the occurrence of thiamine-responsive varieties of maple syrup urine disease.
Biochemical Aspects of Disordered Metabolism
Enzymatic Defect and Essential Consequences
The enzymatic defect in maple syrup urine disease involves the oxidative decarboxylation of the BCKAs. The obvious consequence is a marked elevation in body fluid levels of the BCKAs and the BCAAs. The importance of these accumulated materials in the genesis of the short-term and long-term neurological abnormalities associated with maple syrup urine disease is indicated by the favorable response to diets low in the BCAAs. Available data suggest that both the BCAAs and the BCKAs have deleterious effects on brain and that the precise effect depends in considerable part on the nature of the experimental system examined.
Biochemical Effects of Excess Branched-Chain Amino Acids, Ketoacids, or Both
Neurochemical effects associated with excessive quantities of BCAAs, BCKAs, or both appear to be caused primarily by alterations of brain amino acids and energy failure ( Table 27.2 ). The alterations of amino acids include a marked increase in BCAAs and a depletion of non-BCAAs. The latter depletion results in part because of impaired amino acid transport across the blood-brain barrier caused by the large quantities of competing BCAAs. However, cellular depletion also occurs secondary to the large influx of leucine, which enters brain from blood more readily than any other amino acid. Leucine first enters astrocytes, which surround brain capillaries and is metabolized by the BCAA transaminase to the alpha-ketoacid called alpha-ketoisocaproate (KIC; see Fig. 27.1 ). KIC enters neurons, and a BCAA transaminase, which uses an amino group of glutamate, reaminates KIC to leucine, forming alpha-ketoglutarate, thereby consuming glutamate. The alpha-ketoglutarate becomes available for the aminotransferase of aspartate and thus consumes aspartate. The result of this process is a diminution in the malate-aspartate shuttle for providing reducing equivalents to the mitochondrion. The consequence is diminished function of the electron transport chain coupled with a direct effect of BCAAs on the chain and on creatine kinase. Indeed, experimental studies of the effects of BCAA on energy metabolism in the cerebral cortex have shown that all the BCAAs reduced energy metabolism and inhibited respiratory chain activity. This effect on respiratory chain activity was prevented by alpha-tocopherol and creatine, suggesting a role for the involvement of free radicals. The disturbance in mitochondrial metabolism results not only in energy failure but also in impaired pyruvate metabolism and increased lactate (see later).
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The principal consequences of the altered amino acids and energy failure are multiple (see Table 27.2 ). Consequences of the amino acid abnormalities include alterations of neurotransmitters derived from amino acids (i.e., reduced gamma-aminobutyric acid [GABA], glutamate, and serotonin). A second effect of the amino acid abnormalities is disturbed protein synthesis, with multiple effects including myelin synthesis (see later). The energy failure likely initiates a cascade to cell death that begins with an increase in cytosolic calcium. The deleterious effects of cytosolic calcium are reviewed in Chapter 13 and include the generation of free radicals, which experimental models of maple syrup urine disease have shown to be involved in cell death. A deleterious calcium-mediated effect on the cytoskeleton also has been shown. The cell edema that is a prominent feature of classic maple syrup urine disease (see later) appears to relate in part to the osmotic effect of the large accumulation of BCAAs, especially leucine, and BCKAs, especially KIC. In addition, recent data suggest that the energy failure results in a failure of the adenosine triphosphate (ATP)-dependent Na + K + ion pump and, as a consequence, cell edema. Cell death is the final result.
Importance of Branched-Chain Ketoacids
Many of the demonstrated deleterious effects of maple syrup urine disease in animal models in vivo and in other systems in vitro have been associated with the branched-chain keto acids. Of these, the ketoacid of leucine (i.e., KIC) is the most critical (see Fig. 27.1 ). Thus clinical neurological deficits in human infants are correlated best with leucine administration or with blood leucine levels (KIC not measured directly). Of the branched-chain ketoacids, only KIC inhibits myelination in cultures of cerebellum. Indeed, other adverse effects described in Table 27.2 involving energy failure, free radical generation, cytosolic calcium accumulation, cytoskeletal disturbance, and cell death have been shown in experimental models particularly or exclusively with KIC. If the ketoacids and especially KIC are critical endogenous toxins, this will have major implications for brain because the transamination of the BCAAs is particularly active in brain (unlike the decarboxylation of the alpha-ketoacids) and would facilitate formation of the ketoacids at the site of greatest sensitivity.
Clinical Features
Of the five types of maple syrup urine disease (lassic, intermediate, intermittent, thiamine-responsive, and lipoamide dehydrogenase deficiency), the classic variety consistently manifests in the newborn period. The onset is in the latter part of the first week and is characterized by poor feeding, vomiting, and stupor ( Table 27.3 ). Abnormalities of tone appear; initial fluctuations between hypotonia and hypertonia are followed quickly by dystonic posturing. Opisthotonos, jaw rigidity, and dysphagia become apparent. Seizures occur in approximately half of the symptomatic infants. The characteristic odor of maple syrup may not be present in the early neonatal period. Cerumen is the best source of the odor. Approximately half of infants exhibit a bulging anterior fontanel and signs of increased intracranial pressure. If the disease is not recognized and treated appropriately, death in the first weeks of life is common. The disorder is more fulminating and malignant than phenylketonuria. In phenylketonuria the clinical presentation is usually delayed for several weeks and is insidious in onset, perhaps because the ketoacids of phenylalanine metabolism are derived from a minor pathway, whereas the ketoacids of BCAA metabolism are derived from the major metabolic pathway.
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Interesting and helpful clinical signs in acute maple syrup urine disease are ocular abnormalities . These have consisted of fluctuating ophthalmoplegias, including internuclear ophthalmoplegia. Ophthalmoplegia may be total, and oculocephalic and oculovestibular reflexes may be absent. In addition, we have seen two infants with maple syrup urine disease who had opsoclonus. Fluctuating ophthalmoplegias and related eye signs should always raise the possibility of a serious metabolic encephalopathy in the newborn period. These findings are not confined to maple syrup urine disease; similar observations have been made in nonketotic hyperglycinemia (see later). The ocular abnormalities may be associated with signs of lower cranial nerve dysfunction, including facial diplegia, absent gag reflex, and weak cry. This constellation of ocular and other cranial nerve signs is often initially mistaken for hypoxic-ischemic encephalopathy or a myopathic disorder.
Neurodiagnostic Studies
The diagnosis of maple syrup urine disease is made on the basis of clinical and metabolic features, but neurodiagnostic studies of value include electroencephalography (EEG) and brain imaging. The EEG during the first 2 weeks demonstrates a characteristic comb-like rhythm, consisting of bursts and runs of 5 to 7 Hz, and primarily monophasic negative activity in the central and central-parasagittal regions during both wakefulness and sleep, especially quiet sleep ( Fig. 27.2 ). The abnormality disappears by 40 days after the initiation of dietary therapy. This rhythm may be present on a background of burst suppression, which also disappears after the onset of therapy. This rhythm on the EEG differs from the alpha and theta bursts of normal infants (see Chapter 10 ) in their presence during both wakefulness and sleep; in neurologically normal infants, the bursts are present only in quiet and transitional sleep. Because of the prominent involvement of brain stem (see later), brain stem auditory evoked responses show impaired brain stem latencies (between waves 1 and 5) with a normal wave 1.
Brain imaging techniques of value in evaluating the infant with maple syrup urine disease include cranial ultrasonography and especially, magnetic resonance imaging (MRI). Cranial ultrasonography, often of minimal value in acute neonatal metabolic disorders, shows increased echogenicity in periventricular white matter, basal ganglia, and thalami as well as by imaging through the squamosal temporal window in brain stem. Used previously, computed tomography (CT) shows decreased attenuation, especially in cerebral white matter and deep nuclear structures ( Fig. 27.3 ). MRI, especially diffusion-weighted MRI, is most valuable. The consistent abnormality on T2-weighted images is symmetrical hyperintensity in cerebellar white matter, dorsal brain stem, cerebral peduncles, thalamus, posterior limb of the internal capsule, globus pallidus, and perirolandic cerebral white matter. Still more striking than findings on T2-weighted images, diffusion-weighted MRI shows a striking increased signal (decreased diffusion) in the same areas (see Fig. 27.3 ). The diffusion values are reduced by 70% to 80%. The abnormality is reversible with prompt treatment of the metabolic disorder. However, a subsequent abnormal signal indicative of abnormal myelin is a sequela, and overt volume loss is noted in infants not effectively or promptly treated. The diffusion-weighted MRI findings are consistent with cytotoxic edema , particularly affecting myelinated regions. The findings are consistent with the neuropathology (see later). Principal abnormalities on MR spectroscopy include elevated lactate as well as elevated BCAAs and BCKAs, consistent with the adverse effects of the latter on energy metabolism (see earlier) (see Fig. 27.3 ).
Genetics
Genetic data indicate autosomal recessive inheritance. Thus familial occurrence, affected male and female infants who are products of consanguineous marriages, and biochemical investigations indicating heterozygosity in parents have been documented. The molecular genetic data thus far do not show a straightforward correlation between genotype and phenotype. Most cases of neonatal onset have involvement of the E1 catalytic component (i.e., the thiamine pyrophosphate–dependent decarboxylase). An exception to the variation in molecular defects in general populations, in which the incidence of the disease is 1 in 185,000 newborns, is the single mutation in nearly all Mennonite cases in the United States, in which the incidence is 1 in 176 newborns.
Metabolic Features
The major metabolic correlates of maple syrup urine disease are metabolic acidosis, branched-chain aminoacidemia and aminoaciduria, branched-chain ketoacidemia and ketoaciduria, and hypoglycemia (see Table 27.3 ). Hypoglycemia appears in approximately 50% of the affected infants.
As indicated earlier, the enzymatic defect involves the oxidative decarboxylation of the BCKAs (see Fig. 27.1 ), which causes the accumulation of the BCAAs and BCKAs. This enzymatic defect can be identified in fresh leukocytes and cultured skin fibroblasts or lymphocytes for diagnosis.
The genesis of the secondary metabolic defects appears to be related principally to the massive accumulation of BCAAs and BCKAs, especially leucine and KIC, its alpha-ketoacid. The ketoacids result in the ketoacidosis, and hypoglycemia is thought to relate principally to the accumulation of leucine. The precise mechanism of the hypoglycemia seen in this disorder is probably multifactorial; a deficiency in gluconeogenic substrates, especially alanine, may be most important. A contributory role of leucine in increasing insulin secretion seems possible but is unproven (see Chapter 25 ).
Neuropathology
The neuropathological features vary with the onset and severity of disease, the type of therapy, and the age at death. Several general conclusions seem warranted. The younger infant may exhibit a slightly enlarged and edematous brain. Neuronal changes are minimal and nonspecific. The most prominent parenchymal disturbance involves myelin and consists of vacuolation ( spongy state ). This last abnormality is most marked in the youngest patients, especially in regions of white matter that myelinate rapidly and near the time of active disease. Older patients show a diminution of myelin. A reduction of oligodendrocytes parallels the extent of myelin deficiency. Signs of myelin breakdown are minimal or absent. Because a similar progression from myelin vacuolation to disturbed myelin deposition is seen in several mutant mice with metabolic defects in myelin formation, it has been considered that the major brain defect observed in maple syrup urine disease and related states (e.g., nonketotic hyperglycinemia, phenylketonuria, and ketotic hyperglycinemia) involves myelin formation. It is likely that such a myelin defect in metabolic disorders could be caused by disturbances of the synthesis of myelin lipids (e.g., certain fatty acids, as in ketotic hyperglycinemia) or of myelin proteins (as in the amino acid disorders).
The chemical correlates of the neuropathological findings are diminutions in the levels of myelin lipids as well as myelin proteolipid protein ( Table 27.4 ). The neuropathological and chemical findings of disturbed myelination and the later evidence of such disturbance on MRI scans are less apparent or absent in patients treated from early infancy (see Table 27.4 ).
| AGE OF INFANT | PERCENTAGE OF CONTROL OF | ||
|---|---|---|---|
| TOTAL LIPID | CEREBROSIDES | PROTEOLIPID PROTEIN | |
| 16 days | 90 | 50 | 67 |
| 25 days | 66 | — | 64 |
| 20 months | 82 | 66 | 57 |
| 36 months a | 81 | 93 | 79 |
a Treated with diet low in branched-chain amino acids from 35 days of age.
An additional neuropathological feature involves neuronal development and consists primarily of deficiencies in dendritic development and in quantities of dendritic spines, sites of synaptic contacts ( Fig. 27.4 ). Additional abnormalities included aberrant orientation of cerebral cortical neurons. Neuronal loss, although not a prominent feature of this disease, is usually apparent in cerebellar granule cells.
Management
Prevention
Prenatal diagnosis and prevention of maple syrup urine disease by therapeutic abortion are well-established approaches. Cultured cells derived from chorionic villus biopsy have allowed diagnosis in the first trimester of gestation.
Early Detection
After birth of an affected child, early detection is critical. Institution of aggressive therapy at 5 days of age or less with close monitoring of leucine levels has been followed by normal intellectual outcome (see earlier discussion). On the other hand, institution of therapy after 14 days of age is very uncommonly followed by normal intellectual development. Distinction from other causes of metabolic acidosis in the neonatal period is important (see Chapter 28 ). The early clinical features and odor of maple syrup, especially in cerumen, are most helpful in making the clinical diagnosis. Neonatal blood screening by tandem mass spectrometry to quantify amino acids in whole blood filter paper specimens is highly sensitive, accurate, and rapid and is the currently preferred approach.
Acute Therapy
Acute episodes are managed by correcting dehydration, lowering toxic levels of BCAAs and BCKAs, limiting protein catabolism, and promoting protein anabolism. A combination of enteral and parenteral therapy is used, including high-dose intravenous thiamine. Intravenous dextrose and intralipid are useful to prevent further protein catabolism, and branched-chain amino acid–free parenteral and enteral preparations help diminish leucine levels promptly. Hemodialysis can be lifesaving. Continuous hemofiltration by a pump-assisted, high-flow venovenous system may be as effective and more convenient, albeit somewhat slower than conventional intermittent hemodialysis. Signs of brain edema have been managed with mannitol, furosemide, and intravenous sodium supplementation to replace urinary sodium losses and maintain a serum sodium level greater than 140 mg/L.
Long-Term Therapy
Subsequent therapy includes a diet that initially contains no BCAAs. Control of plasma leucine levels is especially crucial, and the adequacy of this control correlates with intellectual outcome in infants with classic maple syrup urine disease. More recent approaches include optimizing specific amino acids (e.g., phenylalanine, tyrosine, tryptophan, histidine, methionine, threonine) that compete with BCAAs for entry into brain via a common transporter (LAT1), providing glutamine, glutamate, and alanine to replenish episodic depletion caused by reverse transamination and correcting deficiencies of omega-3 essential fatty acids, zinc, and selenium in a special formula. Close supervision is mandatory because relapses may occur with minor infections or for no apparent reason. A more favorable outcome is related particularly to early onset of therapy, careful biochemical monitoring, and early introduction of natural foods to provide adequate nutrition, especially protein anabolism. a
a References .
When dietary therapy is instituted before the onset of symptoms (detected because of an earlier affected sibling), a normal neurological outcome can be achieved. In infants who develop symptoms, the time of detection and institution of therapy are very important. In one earlier study, those detected and treated at 5 days of age or less had a mean intelligence quotient (IQ) of 97 ± 13 versus 65 ± 20 in those detected and treated at 6 or more days of age. In a more recent study, with particularly vigorous metabolic care, most infants with onset of therapy in the second week had favorable neurological outcomes.
Nonketotic Hyperglycinemia (Glycine Encephalopathy)
Nonketotic hyperglycinemia is an inborn error of metabolism in which large amounts of glycine accumulate in body fluids and a serious neonatal neurological disorder occurs. The disturbance involves the cleavage of glycine to carbon dioxide and a one-carbon fragment. This disorder is approximately twice as common as ketotic hyperglycinemia (see Chapter 28 ), from which it should be distinguished. Because nonketotic hyperglycinemia involves the central nervous system directly, the term glycine encephalopathy may be more appropriate.
Normal Metabolic Aspects
Glycine, the simplest of amino acids, is nonessential because it can be synthesized in numerous ways in humans. It is abundant in most proteins and, indeed, approximately 50% of ingested glycine is involved in the synthesis of protein ( Fig. 27.5 ). In addition, however, a large portion of glycine is converted to serine, which, in turn, is involved in the synthesis of phospholipids as well as oxidation to carbon dioxide through the citric acid cycle (see Fig. 27.5 ). Glycine is also cleaved to a one-carbon fragment that is then used in a wide variety of synthetic reactions. In addition, glycine is the precursor of such other critical compounds as purines, glutathione, and porphyrins.
The major roles of glycine as a neurotransmitter are almost certainly crucial for the neurological features of nonketotic hyperglycinemia. It is now clear that glycine has two neurotransmitter roles in the central nervous system, one inhibitory and one excitatory, that are influenced by maturation ( Table 27.5 ). The classic glycine receptor is inhibitory and located primarily in spinal cord and brain stem. This receptor is inhibited by strychnine. However, like the GABA A receptor, it appears to be excitatory during early brain development in animal models. The basis of such early excitatory characteristics may be similar to that for the early excitatory GABA A receptors (see Chapter 12 ); thus the immature brain appears to have increased intracellular chloride because of delayed maturation of the chloride exporter. The result would be chloride efflux and depolarization (excitation) rather than chloride influx and hyperpolarization (inhibition) when glycine activation of its receptor opens the chloride channel. Whether all or a portion of these classic glycine receptors is also excitatory in the human newborn brain is unknown. The possibility that both inhibitory and excitatory receptors are present in the brain stem is suggested by the frequency of both apnea and hiccups in newborns with nonketotic hyperglycinemia. In addition, glycine acts at a second receptor site associated with the N -methyl- d -aspartate (NMDA) receptor-channel complex and potentiates the action of glutamate at this receptor. This receptor is located throughout the central nervous system, including cerebrum and cerebellum. Thus glycine acting at this second receptor is excitatory and indeed can lead to glutamate-induced excitotoxic neuronal death (see later). The excitation may be reflected clinically in the recalcitrant seizures in newborns with nonketotic hyperglycinemia. Because the neonatal nervous system is particularly sensitive to NMDA receptor–mediated neuronal death (see Chapter 13 ), it is clear that characteristics of the immature central nervous system cause glycine to be both excitatory and neurotoxic. In addition, the binding of glycine to the NMDA receptor increases postnatally in human cerebral cortical neurons by 100% from term to 6 months. These issues are directly relevant to the clinical, neuropathological, and therapeutic aspects of nonketotic hyperglycinemia (see later discussions).
| GLYCINE RECEPTORS | ||
|---|---|---|
| CLASSIC | NMDA | |
| Major sites in central nervous system | Spinal cord, brain stem | Diffuse, including cerebral cortex, basal ganglia, cerebellum |
| Primary action | Inhibitory | Excitatory |
| Mechanism of action | Opens chloride channel | Potentiates activation of NMDA receptor by glutamate |
| Developmental feature | Excitatory early in brain development (?) | Most abundant early in development |
| Antagonist | Strychnine | NMDA antagonist (MK-801), glycine site antagonist (HA-966) |
| Potential clinical correlates | Respiratory failure, weakness, hypotonia | Seizures, myoclonus, neuronal toxicity |
Biochemical Aspects of Disordered Metabolism
Enzymatic Defect and Essential Consequences
The enzymatic defect in nonketotic hyperglycinemia involves the glycine cleavage enzyme system, which converts the C 1 of glycine to carbon dioxide and results in the formation of a hydroxymethyl derivative of tetrahydrofolate, the key one-carbon donor ( Fig. 27.6 ). A potential particular importance of the glycine cleavage system in early brain development is suggested by the finding of threefold to fivefold higher activities in brain of the first-trimester fetus than in brain of the adult. The enzyme is expressed early in development in neural stem/progenitor cells in the germinative zones and then later in radial glial cells. Moreover, glycine receptors in developing cerebrum are important in early neuronal development and differentiation. These considerations could partly explain the disturbances in axonal and later myelin development that likely underlie the defects in corpus callosum observed in nonketotic hyperglycinemia (see later).
Abnormalities of two of the four component proteins of the glycine cleavage enzyme complex (i.e., P-protein [the pyridoxal-dependent decarboxylase] encoded by the GLDC gene and T-protein [a tetrahydrofolate-requiring component] encoded by the AMT gene) have been identified as the molecular abnormality in the severe neonatal cases. In one study of 30 cases, 87% exhibited a defect of the P-protein, and the remainder had a defect of the T-protein. These data clarified the earlier in vivo observations in affected infants of defects in the formation of carbon dioxide from the C 1 of glycine and in the formation of the C 3 of serine from the C 2 of glycine (see Fig. 27.6 ).
This aminoacidopathy is distinctive in that the enzymatic defect has been shown to occur in brain , a
a References .
and, indeed, this fact is probably critical in the pathogenesis of the functional and structural features of the disorder. The immediate result is markedly elevated brain concentrations of glycine. Hyperglycinemia from other causes, including ketotic hyperglycinemia, is not associated with elevated levels of glycine in brain or with disturbance of glycine cleavage in brain. Several lines of evidence suggest that the presence of the defect in brain and the resulting accumulation of glycine in brain are critical in the neurotoxicity. First, a deficiency of the product of the glycine cleavage reaction (i.e., the one-carbon tetrahydrofolate derivative) is not likely to be highly important because this compound can be generated by other pathways. Second, administration to very young patients with nonketotic hyperglycinemia of sodium benzoate, which is effective in lowering the plasma glycine level (through the formation of a water-soluble excretable conjugate) but not the cerebrospinal fluid (CSF) glycine level, does not have consistently beneficial neurological effects (see later). Third, strychnine, a centrally acting antagonist of glycine, is effective in improving certain aspects of the neurological status of at least some affected patients (see later discussion).Biochemical Effects of Excessive Brain Glycine
The mechanism of the deleterious effect of glycine on neurological function may relate to glycine’s neurotransmitter roles. Both inhibitory and excitatory actions likely occur. Concerning inhibition, the classic inhibitory glycine receptor may account in part for the apparent suppression of ventilation through action on the brain stem neurons crucial for respiratory drive as well as for the hypotonia and weakness through action on spinal cord neurons. Concerning excitation, three factors may be relevant. First, as noted earlier, early in brain development some classic inhibitory glycine receptors may be excitatory. Hiccups, which likely represent a brain stem excitatory effect, may relate to paradoxically excitatory glycine receptors (see earlier). Second, any existing inhibitory glycine receptors could exhibit desensitization with persistent exposure to high concentrations of glycine. Indeed, experimental evidence indicates that excess glycine may result in a desensitization of glycine receptors at the postsynaptic membrane, which would result in diminished inhibition of certain pathways. Third, probably the most potent excitatory influence of glycine is exerted at the NMDA receptor, as described earlier. The result of these excitatory influences could include seizures, hyperexcitability, and myoclonus.
The mechanism of the deleterious effect of glycine on neural structure may relate to a disturbance in myelin proteins and to excitotoxic neuronal effects. Neuropathological observations demonstrate a striking myelin disturbance in nonketotic hyperglycinemia, similar to that observed with maple syrup urine disease and other aminoacidopathies. Because protein synthesis is disturbed when one amino acid is present in markedly abnormal quantities, one possibility is that excessive brain glycine leads to the myelin disturbance by causing a defect in the synthesis of one or more myelin proteins. In addition, neuronal loss in cerebrum and cerebellum may be excitotoxic (see later discussion). Disturbances in cerebral development, of prenatal origin (see later), may relate to both the deficient action of the glycine cleavage enzyme and the excessive action of glycine on glycine and NMDA receptors. Thus, as noted earlier, the glycine cleavage system is important in developing neuroepithelium in stem/progenitor cells and radial glial cells. The action of glycine on both the glycine and NMDA receptors is important in brain development; in excess these actions could be deleterious. One such deleterious effect could be excitotoxicity mediated by the NMDA receptor.
Clinical Features
The onset of nonketotic hyperglycinemia in the typical case is in the first days of life, most commonly the first 2 days of life, with ineffective suck, impaired ventilatory effort (or apnea), stupor, hypotonia, seizures, multifocal myoclonus, and hiccups ( Table 27.6 ). Approximately two thirds of infants exhibit the onset before 48 hours of life; onset in the first hours of life is not unusual, and abnormal fetal movements suggestive of myoclonus or hiccups have been observed. Seizures occur on the first postnatal day in approximately 15%, by day 3 in nearly 50%, and by day 30 in approximately 70% of patients. Hiccups are a particularly helpful and frequent clinical sign. A mother of one of the affected newborns volunteered before the diagnosis was suspected that she felt that her fetus experienced frequent hiccups. It is important to specifically ask the mother about such fetal movement. As with maple syrup urine disease, interesting and useful neurological signs include a variety of ophthalmoplegias, which may be fluctuating in character. Of particular importance is the need for mechanical ventilation in approximately two thirds of patients. Rapid evolution of intractable seizures, stimulus-sensitive myoclonus, and coma is common.
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The neonatal EEG is abnormal in at least 90% of infants. The most common finding is the burst-suppression pattern, and nonketotic hyperglycinemia is the most common metabolic cause of the syndrome of early myoclonic encephalopathy (myoclonic seizures, burst-suppression EEG; see Chapter 12 ). Brain stem auditory evoked responses are characterized by delayed brain stem conduction times (e.g., wave I to V latency).
Brain imaging is notable in the neonatal period for the relatively frequent findings of agenesis or hypoplasia of the corpus callosum and abnormalities of cerebral white matter, with subsequent evidence for hypomyelination and to a lesser extent, cerebral cortical atrophy. The CT scan may demonstrate decreased attenuation of the cerebral white matter ( Fig. 27.7A ) and partial or complete agenesis of the corpus callosum. MRI is superior to CT in demonstrating these features (see Figs. 27.7B and 27.8 ) and is the recommended neuroimaging modality. Thus, decreased attenuation of cerebral white matter may be observed, but, more strikingly, on diffusion-weighted MRI, one sees increased signal (decreased diffusion) in dorsal brain stem, cerebral peduncles, and posterior limbs of the internal capsule (see Fig. 27.8 ). These features are consistent with the vacuolating myelinopathy observed at neuropathological examination (see later), as is also seen with maple syrup urine disease (see earlier). The abnormalities of corpus callosum are best visualized in vivo by MRI and occur in nearly 50% of newborns with severe disease. a
a References .
Progression of findings to abnormal signal and then atrophy of cerebral white matter and, to a lesser extent, cerebral cortex is common. MR spectroscopy shows a striking increase in brain glycine levels, consistent with the locus of the enzymatic defect (see earlier) ( Fig. 27.9 ). In conventional short-echo spectra, glycine cannot be distinguished from the normal myoinositol peak; with long-echo spectra, the elevation of glycine is seen clearly (see Fig. 27.9 ).
A syndrome of transient neonatal nonketotic hyperglycinemia, which clinically can be indistinguishable from the better-known classic neonatal form first described, has been elucidated. The clinical presentation has been characterized by onset of seizures in the first days of life with hypotonia and depressed level of consciousness; one infant exhibited coma and respiratory failure. All infants survived, and six of eight were normal neurologically on follow-up. The diagnosis was made by the finding of increased concentrations of glycine in the CSF, urine, and plasma, with the most consistent elevation in the CSF. The metabolic abnormalities disappeared within 2 to 8 weeks. A transient defect in the glycine cleavage enzyme is presumed but has not been documented. The existence of this syndrome with a markedly better outcome than that associated with the more typical persistent form raises difficult ethical issues in the management of classic nonketotic hyperglycinemia. In the latter disorder, cessation of life support is often considered in the severely ill infant early in the clinical course because of the very poor prognosis despite the typical occurrence of recovery of ventilatory function later in the neonatal period.
Clinical distinction of transient nonketotic hyperglycinemia from atypical variants of nonketotic hyperglycinemia is also difficult. These infants present clinically like those with classic nonketotic hyperglycinemia, and the outcome has ranged from normal neurological status to death early in infancy. The distinction of this milder variant from transient neonatal nonketotic hyperglycinemia is based on resolution of the metabolic defects in the latter but not fully in the former. Moreover, the milder forms have been shown to be associated with considerable (20% to 30%) residual glycine cleavage enzyme activity. Decisive distinction from true transient disease requires determination of molecular genetic analysis of the glycine cleavage system.
The outcome of the severe neonatal form of nonketotic hyperglycinemia has been generally poor. Overall approximately 30% to 35% of infants die, often in the neonatal period, and most survivors have serious neurological disturbances, including severe developmental failure, recurrent seizures, and severe abnormalities (e.g., hypsarrhythmia) on the EEG. In one recent series only 25% of infants evaluated at 15 months were able to smile, 4%, to sit alone, and none to babble or speak words. Infants who present in the neonatal period or very early infancy, generally without severe signs at onset, have a less dire outcome but do exhibit notable gender differences in outcome ( Table 27.7 ). Thus, in one series of 65 infants, although overall 12% died in the neonatal period, the gender-specific mortality rates were 28% for female patients and 0% for male patients. Indeed overall median age at death was 2.6 years for male patients versus less than 1 month for female patients. The male advantage was noted also for outcome in survivors. Of survivors 3 years or older, although severe deficits occurred overall in 60%, gender-specific rates of poor outcome were 100% for female patients and 29% for male patients. Of the original 65 infants, 10 infants (15%) could walk and say or sign words, and these were all male. None of these 10 were neurologically normal, however. In a recent mixed series of 124 cases of nonketotic hyperglycinemia with onset in the neonatal period and early infancy, the best predictors of poor outcome were CSF glycine level greater than 230 µM, markedly elevated CSF/plasma glycine ratio (median 0.22), and genetic mutations expected to allow no residual activity. Therapeutic intervention may modify the unfavorable outcome in nonketotic hyperglycinemia (see later).
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Distinction From Ketotic Hyperglycinemia
It is important to distinguish nonketotic hyperglycinemia from ketotic hyperglycinemia, particularly in view of the observation that not all patients with ketotic hyperglycinemia exhibit consistent ketosis. Early therapeutic intervention may be particularly beneficial in ketotic hyperglycinemia. Although this is not yet clearly the case with severe nonketotic hyperglycinemia, some observations raise the hope that specific therapy will become available (see later). Features helpful in the distinction of nonketotic and ketotic hyperglycinemia are included in Table 27.8 .
| NONKETOTIC | KETOTIC | |
|---|---|---|
| Severe neonatal illness | + | + |
| Seizures | + | + |
| Hiccups | + | – |
| Ketoacidosis | – | + |
| Neutropenia-thrombocytopenia | – | + |
| Primary defect in glycine metabolism | + | – |
| Dietary therapy effective | – | + |
Genetics
Genetic data indicate autosomal recessive inheritance. Thus familial occurrence, parental consanguinity, and intermediate molecular defects in heterozygotes have been recorded. More recent data show extensive intragenic molecular heterogeneity in classic NKH, including 78 novel mutations in GLDC and 18 novel mutations in AMT. In particular, nonsense mutations, frameshift mutations, exonic deletions, and duplications, which result in no residual activity, were more frequent in patients with severe neurodevelopmental outcomes than in those with attenuated outcomes.
Metabolic Features
The major biochemical correlate of nonketotic hyperglycinemia is marked accumulation of glycine in blood, urine, and CSF. Particularly characteristic is the accumulation of glycine in the CSF. Values generally range between 85 and 280 µmol/L, with control subjects generally having values less than 10 µmol/L. a
a References .
In a recent large series, the median value among severely affected newborns was 213. The ratio of the concentration of glycine in CSF to that in plasma, an important diagnostic measure, generally ranges from 0.09 to 0.25 µmol/L, with control values approximately 0.02 µmol/L. The median ratio in a recent large series in severely affected newborns was 0.22. This pronounced elevation of CSF glycine level is not observed in other varieties of hyperglycinemia and presumably relates to the presence of the enzymatic defect in brain in the patients with nonketotic hyperglycinemia. As noted earlier, a distinct correlation exists between the degree of elevation of the ratio of CSF glycine to plasma glycine and the severity of the clinical phenotype. The defect in the glycine cleavage reaction (see Fig. 27.6 ) in brain, liver, and probably other tissues adequately explains the accumulation of glycine in all body fluids. As noted earlier, elevated levels of glycine in the brain of living infants with the disease have been demonstrated by MR spectroscopy.Neuropathology
Neuropathological findings from studies of more than 20 infants have been described. a
a References .
The dominant abnormality has involved myelin, and the nature of the disturbance is similar to that noted for ketotic hyperglycinemia, maple syrup urine disease, and various other aminoacidopathies. The essential features are vacuolation of and diminution in myelin ( Fig. 27.10 ). No striking involvement of neurons or sign of myelin breakdown is noted. Vacuolation is more common in younger patients, and myelin diminution is more common in older patients, findings suggesting that myelin formation is deranged and that the early sign of this derangement is vacuolation. Ultrastructural studies support the notion of origin of the vacuoles from newly formed myelin sheaths ( Fig. 27.11 ). Involvement is greatest in those systems that myelinate around the time of birth. A prenatal onset of the process is supported by the frequent finding of partial or total agenesis of the corpus callosum (see earlier discussion). MRIs have also shown abnormalities of gyral development in occasional cases. Whether such abnormalities could be related to the expression during development of the glycine cleavage system in radial glial cells (see earlier) is an intriguing possibility. More detailed MRI studies will be of interest.
Neuronal injury in cerebrum has not been described consistently at autopsy. The possibility of excitotoxic neuronal injury initiated at the NMDA receptor (see earlier discussion) has not been defined clearly, although this occurrence seems likely. As noted earlier, apparent cerebral cortical atrophy is a common feature on brain imaging studies in infants who survive beyond the neonatal period.
Management
Prevention
Demonstration of very low or no activity of the glycine cleavage system in biopsy samples of chorionic villus has allowed diagnosis in the first trimester. On the basis of such prenatal identification, prevention by therapeutic abortion has been carried out.
Early Detection
Early detection is important because institution of therapy in the neonatal period provides the best opportunity to ameliorate, albeit only partially, the very unfavorable neurological outcome (see later discussion). A serious ethical issue arises with the severely affected infant who requires ventilatory support. With the unfavorable prognosis characteristic of this disease, the decision to discontinue life support frequently arises. Because the severe respiratory failure often resolves later in the neonatal period (see earlier discussion), the continuation of early life support may result in the recovery of ventilatory function but a very poor neurological outcome. With the recognition of transient nonketotic hyperglycinemia, however, the decision to discontinue ventilatory support early in the neonatal period in infants with nonketotic hyperglycinemia is especially difficult. Moreover, recent therapeutic attempts provide some reason for hope in this disorder (see next section).
Therapeutic Attempts
The three fundamental aims of therapy in this disorder are to (1) lower tissue glycine levels, (2) treat seizures, and (3) ameliorate excitotoxicity at the NMDA receptor. The available interventions have attained these aims with reasonable success for the first, with moderate success for the second, and with limited success for the third.
Sodium Benzoate.
Sodium benzoate has been used to lower glycine in blood because an amide bond between glycine and benzoate is formed and the resulting hippuric acid is excreted. The plasma glycine levels are reduced to near normal but, unfortunately, CSF glycine levels are often not similarly affected. a
a References .
However, with doses as high as 750 mg/kg per day, a substantial decline in CSF glycine levels has been effected and a decrease in seizures reported. Moreover, with doses nearly as high, a decrease in brain (as well as CSF) glycine levels has been shown by MR spectroscopy. High-dose benzoate therapy may result in carnitine deficiency, and carnitine supplementation may be necessary. Unfortunately, however, thus far no beneficial effect on cognitive development has been reported with benzoate therapy.Strychnine.
The initial therapeutic approach directed at the effects of glycine in the central nervous system was the use of strychnine. a
a References .
The rationale for administration of this drug is its role as a specific antagonist of the inhibitory glycine receptor at the postsynaptic membrane. In general, severely affected neonatal patients have not had apparent benefit, despite onset of therapy from the first hours or days of life. Nevertheless, some apneic infants have responded to strychnine sufficiently to allow extubation. The principal reason for the lack of major benefit from strychnine presumably relates to the finding that the drug has no effect on glycine’s allosteric activation of the NMDA receptor and thereby excitotoxicity.Benzodiazepines.
A class of agents that acts principally by enhancing GABA receptor inhibitory function, the benzodiazepines, has been used in infants with nonketotic hyperglycinemia. A beneficial response on seizure frequency, often at relatively high doses, has been observed in some patients. However, antiepileptic effects have been inconstant, and no beneficial effect on neurological development has been observed. The latter failure probably relates in part to a lack of effect of benzodiazepines at the NMDA receptor and the concept that GABA A receptors in the newborn may be largely excitatory (see Chapter 12 ).
Excitatory Amino Acid Antagonists.
A theoretically promising therapy in nonketotic hyperglycinemia involves agents that are excitatory amino acid antagonists. b
b References .
In general, the most commonly used agents have been dextromethorphan or ketamine in combination with sodium benzoate. A beneficial effect on seizures has been documented. However, amelioration of cognitive deficits has generally not been achieved. Further improvements in development of NMDA antagonists, with specific action at the glycine site on the NMDA receptor, may lead to more favorable effects on outcome. A recent report of three infants with neonatal nonketotic hyperglycinemia and early myoclonic encephalopathy with poor seizure control using standard pharmacological therapy (dextromethorphan and sodium benzoate) describes a dramatic reduction of seizures and improved quality of life when treatment with a ketogenic diet was instituted. Antenatal therapy with an effective excitatory amino acid antagonist may be necessary for optimal benefit. The findings of hypoplastic corpus callosum, elevated levels of CSF glycine at birth, the absence of glycine cleavage activity in fetal brain, and the occurrence of severe neurological signs in the first hour of life all support this contention (see earlier).Conclusions.
The data just reviewed are disappointing concerning effective therapy of nonketotic hyperglycinemia. Use of sodium benzoate and an NMDA antagonist theoretically is the best current combination. Additional anticonvulsant therapy may be required. Nevertheless, it does not appear that this approach, even if improved with newer agents, will correct all the deficits in this disorder. The reasons for this prediction relate to several factors. First, it is likely that brain injury, maldevelopment, or both occur in utero because of the locus of the enzymatic defect in brain (i.e., a locus unavailable to the benefits of placental function). Second, the role of the disturbance of myelination in the genesis of the intellectual failure and some of the other neurological disturbances in nonketotic hyperglycinemia presumably is largely separate from the neurotransmitter effects of glycine.
Hyperammonemia
Hyperammonemia in the neonatal period may result in serious derangements of neurological function and structure. The Krebs-Henseleit urea cycle is the major pathway of ammonia elimination in mammals; thus defects in the enzymes catalyzing the five steps of this pathway are important causes of hyperammonemia. Neonatal hyperammonemia results from defects of the first four of these five steps. Elevations of blood ammonia levels occur in certain other inborn errors of metabolism and have also been demonstrated in a significant proportion of premature and asphyxiated infants ( Table 27.9 ). In the last two instances, the hyperammonemia is not secondary to an inborn error of ammonia metabolism. (Not listed in Table 27.9 is hyperammonemia with hepatic failure or with total parenteral nutrition, because the severity of the hyperammonemia is rarely marked and clinical phenomena referable to the hyperammonemia are most unusual in these settings.) In the following discussion, the normal aspects of ammonia metabolism, the biochemical aspects of disordered metabolism, and the principal clinical syndromes associated with neonatal hyperammonemia are reviewed. Emphasis is placed not only on the deficits in the urea cycle enzymes but also on the disturbance observed in premature infants. Hyperammonemia associated with perinatal asphyxia is discussed in Chapter 20 .
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Normal Metabolic Aspects
Major Sources and Fates of Ammonia
The major sources of ammonia in mammals are amino acids and purine nucleotides (amino groups of adenine, guanine, and their derivatives). Although small amounts of ammonia are used for the synthesis of certain amino acids (primarily by transamination) and pyrimidines, the principal fate of ammonia is biosynthesis of urea through the urea cycle for waste nitrogen disposal ( Fig. 27.12 ).
Urea Cycle
The urea cycle consists of five steps, the first two of which are catalyzed by the mitochondrial enzymes carbamyl phosphate synthetase and ornithine transcarbamylase and the latter three of which are catalyzed by the cytosolic enzymes argininosuccinic acid synthetase, argininosuccinase, and arginase (see Fig. 27.12 ). An important obligatory positive effector of carbamyl phosphate synthetase is N -acetylglutamate, which is synthesized in the mitochondrion from acetyl-coenzyme A and glutamate. The liver is the only organ that is quantitatively important in urea synthesis. In human neonatal brain, only argininosuccinic acid synthetase is present in significant quantities (i.e., 155% of the hepatic activity), whereas activities of carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinase, and arginase are present in relatively small quantities (3%, 0.2%, 14%, and 2% of the respective hepatic activities).
Ammonia Disposal in Brain
Ammonia is formed constantly in brain and, indeed, ammonia concentrations in brain in adult animals are 60% to 100% higher than in blood. Ammonia in brain is eliminated by diffusion and by conversion to glutamate and, particularly, to glutamine (see Fig. 27.10 ). Glutamine also may diffuse from brain. At least one mode of glutamine transport from brain involves a transporter shared with tryptophan, so glutamine efflux from brain is accompanied by tryptophan influx into brain (see later discussion).
Biochemical Aspects of Disordered Metabolism
Enzymatic Defects and Essential Consequences
Of the major causes of hyperammonemia in the perinatal period (see Table 27.9 ), those studied in most detail involve the enzymes catalyzing the reactions of the urea cycle. These disorders affect the hepatic enzymes and, to a variable extent, the enzymes in other tissues. Hyperammonemia is a prominent consequence and, depending on the site of the enzymatic block, so are aberrations of amino acids in blood or urine. The causes for the striking disturbances in function and structure of the central nervous system observed with these hyperammonemic states are not entirely understood. Mechanisms that are supported by some experimental and clinical evidence are displayed in Fig. 27.13 and discussed in the next sections.
