Amino Acids

Transamination

The first two steps in the degradation of the branched-chain amino acids (BCAAs) are shown in Fig. 31.1 . The initial transamination is thought to occur via a single transaminase. Presently, there are only two genes that encode a BCAA transaminase. The first is BCAT1 . The cBCAT protein resides in the cytoplasm of brain cells and plays a key role in the neuronal-astrocyte shuttle involving BCAAs, glutamate, glutamine, and ammonia. The second gene is BCAT2 . The mBCAT protein resides in the mitochondria and is ubiquitously expressed. The only exception is the liver where there is little or no expression. That exception underlies the reason that all of the AAs, except for the BCAAs , are first metabolized in the liver after a meal. 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 BCKA dehydrogenase complex to the corresponding ketoacids (see Fig. 31.1 ). Oxidative decarboxylation of the three alpha-ketoacids is particularly active in the liver; intermediate in the kidney and heart; and low in muscle, adipose tissue, and the brain. The BCKA dehydrogenase 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 MSUD.

Enzymatic Defect and Essential Consequences

The enzymatic defect in MSUD 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 MSUD 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 the 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 or Ketoacids, or Both

Neurochemical effects associated with excessive quantities of BCAAs, BCKAs, or both, appear to be caused primarily by alterations of brain AAs and by energy failure ( Table 31.2 ). The alterations of AAs include a marked increase in BCAAs and a depletion of non-BCAAs. The latter depletion results in part because of impaired AA transport across the blood-brain barrier caused by the large quantities of competing BCAAs. However, cellular depletion occurs also secondary to the large influx of leucine, which enters the brain from the blood more readily than any other AA. Leucine first enters astrocytes, which surround brain capillaries, and is metabolized by the BCAA transaminase to the alpha-ketoacid, alpha-ketoisocaproate (KIC; see Fig. 31.1 ). 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 the latter process is a diminution in the malate-aspartate shuttle for providing reducing equivalents to the mitochondrion. The consequence is diminished function of the election transport chain, coupled with a direct effect of BCAAs on the chain and on creatine kinase. Indeed, experimental studies of the effects on BCAAs on energy metabolism in the cerebral cortex showed that all the BCAAs reduced energy metabolism and all inhibited respiratory chain activity. This effect on respiratory chain activities was prevented by alpha-tocopherol and creatine, suggesting a role for free radical involvement. The disturbance in mitochondrial metabolism results not only in energy failure but also in impaired pyruvate metabolism and increased lactate (see later).

The principal consequences of the altered AAs and the energy failure are multiple (see Table 31.2 ). Consequences of the AA abnormalities include alterations of neurotransmitters derived from AAs (i.e., reduced gamma-aminobutyric acid [GABA], glutamate, and serotonin). A second effect of the AA 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 16 and include generation of free radicals, shown to be involved in cell death produced by experimental models of MSUD. A deleterious calcium-mediated effect on cytoskeleton also has been shown. Cell edema is a prominent feature of classic MSUD (see later) during acute metabolic decompensation. It may be related in part to the osmotic effect of the large accumulation of BCAAs, especially leucine, and BCKAs, especially KIC. However, the levels of each of these compounds are probably only in the low millimolar range and thus unlikely to reach levels that are osmotically significant. Data suggest that energy failure may be related to impairment 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 in MSUD 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 ( Fig. 31.1 ). Thus clinical neurological deficits in human infants are correlated best with leucine administration or with blood leucine levels (KIC not measured directly), and of the BCKAs, only KIC inhibits myelination in cultures of cerebellum. Indeed, other adverse effects described in Table 31.2 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 the brain because the transamination of the BCAAs are particularly active in the brain (unlike the decarboxylation of the alpha-ketoacids) and would facilitate formation of the ketoacids at the site of greatest sensitivity.

Neurodiagnostic Studies

The diagnosis of MSUD is made on the basis of clinical and metabolic features, but neurodiagnostic studies of value include electroencephalogram (EEG) and brain imaging. The EEG during the first 2 weeks demonstrates a characteristic “comblike” rhythm, consisting of bursts and runs of 5 to 7 Hz, primarily monophasic negative activity in the central and central-parasagittal regions during both wakefulness and sleep, especially quiet sleep ( Fig. 31.2 ). 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 onset of therapy. This rhythm on the EEG rhythm differs from the alpha and theta bursts of normal infants (see Chapter 13 ) 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 brainstem (see later), brainstem auditory-evoked responses show impaired brainstem latencies (between waves 1 and 5) with a normal wave 1.

Brain imaging techniques of value in evaluation of the infant with MSUD 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 brainstem. Used previously, computed tomography (CT) shows decreased attenuation, especially in cerebral white matter and deep nuclear structures ( Fig. 31.3 ). MRI, especially diffusion-weighted MRI, is most valuable. The consistent abnormality on T2-weighted images is symmetrical hyperintensity in cerebellar white matter, dorsal brainstem, 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. 31.3 ). 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 magnetic resonance (MR) spectroscopy include elevated lactate and elevated BCAAs and BCKAs, consistent with the adverse effects of the latter on energy metabolism (see earlier) ( Fig. 31.3 ).

Genetics

MSUD is an autosomal recessive disorder due to biallelic pathogenic variants in one of the BCKDHA, BCKDHB , or DBT genes. The severity of phenotype is related to the residual enzyme activity in relation to the amount of BCAA in the diet and the catabolic status of the patient. Most cases of neonatal 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. Carrier testing has been performed in the U.S. Mennonite population in Pennsylvania via the Clinic for Special Children. Genetic counseling is recommended for at-risk individuals and populations to determine the genetic risk.

Antenatal Diagnosis and Prevention

Prenatal diagnosis and prevention of MSUD by therapeutic abortion are well-established approaches. Prenatal molecular genetic testing can determine the disease status in ongoing pregnancies. Preimplantation genetic testing is available before pregnancy.

Early Detection and Diagnosis

Gene sequencing can establish the diagnosis in patients with phenotypes compatible with MSUD or in individuals who are at risk for its presence because of family history. Early detection is critical. Institution of aggressive therapy at 5 days of life or less with close monitoring of leucine levels has been followed by normal intellectual outcome (see earlier). Moreover, institution of therapy after 14 days of life is very uncommonly followed by normal intellect. Distinction from other causes of metabolic acidosis in the neonatal period is important (see Chapter 32 ). The early clinical features and the odor of maple syrup, especially in cerumen, are most helpful in making the clinical diagnosis. Neonatal blood screening by tandem mass spectrometry to quantify AAs 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 BCAA-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 MSUD. More recent approaches include optimizing specific AAs (e.g., phenylalanine, tyrosine, tryptophan, histidine, methionine, threonine) that compete with BCAAs for entry into the 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.

When dietary therapy is instituted before the onset of symptoms (detection because of an earlier affected sibling), a normal neurological outcome can be achieved. As noted earlier, 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 life 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 life. In a more recent study, with particularly vigorous metabolic care, most infants with onset of therapy in the second week had favorable neurological outcomes.

Enzymatic Defect and Essential Consequences

The enzymatic defect in NKH 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. 31.6 ). 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 the brain of the first trimester fetus than in the 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 explain, in part, the disturbances in axonal and later myelin development that likely underlie the defects in corpus callosum observed in NKH (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. Abnormalities in the third component of the glycine cleavage enzyme complex, GCS H-protein component encoded by the GCSH gene has also been proposed. In one study of 578 families, 80% of individuals exhibited a defect in the P-protein due to mutations in the GLDC gene, and the remainder had a T protein defect due to mutations in the AMT gene. With this pathophysiology, affected infants would be expected to have variable formation of carbon dioxide from the C ₁ of glycine and also in the formation of the C ₃ of serine from the C ₂ of glycine ( Fig. 31.6 ).

This aminoacidopathy is distinctive in that the enzymatic defect has been shown to occur in the brain , 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 in the brain can also be caused by defects in the genes that play a role in the biosynthesis of lipoate, which is a cofactor in the glycine cleavage system. In addition, perturbations in pyridoxal-5′-phosphate biosynthesis and glycine transport can also result in hyperglycinemia in the CNS. ) Several lines of evidence suggest that the presence of the defect in the brain and the resulting accumulation of glycine in the 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 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, to very young patients with NKH 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).

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 brainstem 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 brainstem 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 NKH, similar to that observed with MSUD and other aminoacidopathies. Because protein synthesis is disturbed when one AA 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 in 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.

Differential Diagnosis of Hyperglycinemia and/or Elevated Glycine in CSF

Hyperglycinemia can be observed due to nongenetic causes, such as valproate utilization, false positive newborn screening results, liver failure, and glycine loading. It is also observed in transient glycine encephalopathy, as noted earlier. Intracerebral hemorrhage and hypoxic-ischemic injury can also cause transient elevations in CSF glycine. Biochemical causes of hyperglycinemia caused by deficient activity of the glycine cleavage enzyme system include defects in GCS cofactor synthesis and metabolism, such as pyridoxine-dependent epilepsy and lipoate disorders; abnormal regulation of GCS due to disorders of intracellular cobalamin metabolism (cblX); glycine transport defect (GLYT1 encephalopathy); and inhibition of GCS activity due to metabolites accumulated in organic acidurias (i.e., ketotic hyperglycinemia). These rare disorders are discussed in specialized sources.

Distinction From Ketotic Hyperglycinemia

It is important to distinguish NKH 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 NKH, 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 31.7 .

Genetics

NKH is an autosomal recessive disorder due to biallelic pathogenic variants in GLDC or AMT genes. At least to date, no pathogenic variants have been detected in patients with transient NKH. There is extensive intragenic molecular heterogeneity in classic NKH, including 52% missense mutations, 11% nonsense mutations, 10% splice-site mutations, 6% small insertions/deletions (InDels), and 21% exonic copy number variants in the GLDC gene and 67% missense mutations, 3% premature stop mutations, or 8.4% splice-site mutations in the AMT gene. The frequency of NKH is estimated to be around 1:76,000 and the disorder is more frequent in certain populations (see earlier). Genetic counseling is recommended for at-risk individuals.