Nonketotic hyperglycemia: Clinical, biochemical, and therapeutic considerations

GENERAL

CLINICAL

FEATURES

Clinical features and biochemical parameters of 25 cases of nonketotic hyperglycinemia reported in detail in the literature are presented in Table 1. While this is not an exhaustive list, it is a representative collection of early-onset cases. From the data available, the likely mode of inheritance is autosomal recessive. Four reported cases (16%) involved consanguineous parents’* 11*37*38 and one report raised consanguinity as a possibility.16 Moreover, 11 (44%) of the 25 patients had a sibling in whom nonketotic hyperglycinemia was either suggested by infant death2>41’ or demonstrated chemically.14, l6 The incidence of the syndrome has not been estimated, although the infrequency of reported cases indicates that it is quite low. Since the syndrome is being recognized with increasing frequency, it is likely that in the past a significant number of infants with this disorder were not diagnosed, and other affected children from large referral centers may not have been reported. Nonketotic hyperglycinemia is perhaps more common than the organic acidemias which tend to be associated with elevated glycine levels in blood and urine.26 As can be ascertained from Table 1, there is considerable variation in the age at which the infant with nonketotic hyperglycinemia becomes symptomatic-from 1 to 42 days. Rarely, onset is delayed until several months of age.18 Some of this spread undoubtedly reflects variability in the attending physicians’ ability to recognize this disorder as well as in the swiftness with which studies of the affected infant were conducted. In most instances onset occurred in the first week of life, and in 15 of the 25 infants, symptoms became apparent during the first 72 hours. In virtually all patients there were no antenatal signs of pathology, and with one exception,’ gestation and delivery of the affected babies were unremarkable. However, recently cases have been reported in which the diagnosis was made antenatally by determining glycine and serine levels in amniotic fluid,37 or postnatally in the first few hours of life by measuring CSF glytine levels.3g The nature of the presenting symptoms reflects the extent to which nonketotic hyperglycinemia manifests as an insult to the developing CNS. Lethargy and poor feeding were initial expressions in nearly all infants. Apnea was another commonly reported early symptom, usually necessitating intubation and assisted ventilation. Additional correlates include diminished or absent neonatal reflexes and difficulty in eliciting suck, grasp, and Moro reflexes. Abnormalities of deep tendon reflexes and muscle tone were less uniform. While 11 (44%) of the case reports reviewed mentioned hyporeflexia and hypotonia, there were also several reports of 8

N3R 2

Nip NP NP NP NP 2 2 1 1

13 14 14 14 15 16 16 16 17 79

NfR NR NR NR + + +

NR + + + + + + + NR -

NfR

N+R NR NR NR + +

NfR +

I& + + + -

+ + + + +

APNEA DIFFICULTY

++ +

+

2

+ + + + + + 1 NR

I NR

NR rr tt

ii: NR L NR NR NR NR NR

i NR

i

f

NR NR i NR

i Y

NR NR

SUCK

GAG

m

m, myoclonic;

m

Y m

NR

:

m

J

m m m

NR

SEIZURES

NR NR NR t -

N

TNR

f, focal; g, generalized;

t NR 4 NR

ii: NR i NR NR NR NR NR

4

NR NR NR i NR NR NR J NR

REFLEX REFLEX REFLEX

MORO

TNR, tonic neck reflux; = equivocal.

N+R

+

A

i

I

NR

+

+ + + + + + + + +

TONE

LETHARGY

MUSCLE

+ = present;

+ + + + + + + + + + + + + + + + + + + + + + + +

DELAY

DEVELOPMENTAL

AND LABORATORY FINDINGSIN 25 REPORTED CASES OF NONKKTOTIC HYPERGLYCINEMIA*

FEEDING

l.-CLINICAL

lot reported; N, normal; NP! neonatal period; = decreased; t & = intermlttent t or J ; -

2 1 4 2 42

.l

1 8 3 3

2 3 4 5 6 7 8 8 8 9 10 11 11 11 12

REFERENCE (days)

AGE AT ONSET

TABLE

-

t

ill NR NR NR + + + + + NfR NR NR + + + + + + = absent;

NfR NR NR NR NR NR NR

+

= in-

2

NR + +

+ + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + +

CSF

Urine

Blood

INCREASEDGLYCINE IN

both muscle tone and tendon reflexes being either exaggerated or variable.13-l5 Seizures, usually generalized or myoclonic, were another constant feature. Focal seizures are rarely reported. Gross dysmorphic features are usually not part of the clinical picture. Additional clinical features mentioned in isolated case histories included fever, bronchopneumonia, microcephaly, and renal failure. 3, ‘9 13*‘* The rarity of such reports suggests that these are incidental findings. CLINICAL

OUTCOME

In terms of mortality and morbidity, the prognosis of nonketotic hyperglycinemia is dismal. In patients that were studied at length, death occurred during the first months7 or even days’, lo of life. Severe global developmental delay tends to affect children who survive infancy. Nyhani reports the fate of Gerritsen’s patient, perhaps the longest survivor, who at 5 years of age was immobile, confined to a hyperextended position, and completely lacked social and adaptive behaviors. PATHOLOGIC

FEATURES

Concerning the associated neuropathology, Shuman and coworkers*’ reported that in nonketotic as well as ketotic hyperglycinemia there are several prominent alterations of brain morphology. Patients dying in the first few weeks of life tend to have vacuolation of myelin in the tracts that myelinate after birth, as well as in those that myelinate most rapidly. These include the extrapyramidal tracts, medial lemniscus, superior cerebellar peduncle, posterior columns, posterior limb. of the internal capsule, and ascending and descending fibers of the brain stem tegmentum. In two children who succumbed after 2 years of age, this vacuolation extended to the optic tracts and chiasm and was accompanied by a diffuse and symmetric deficiency of cerebral white matter. Atrophic peripheral nerves similar to those found in other amnioacidopathies were also reported by Shuman and co-workers.*’ Since synthesis of a given protein depends on the presence of specific amino acids,*i these neuropathologic changes presumably reflect inhibition of myelin-synthesizing ability due to an altered cytoplasmic amino acid composition. Similar conclusions resulted from the pathologic studies of Brun et al., who documented vacuolation and spongy degeneration of white matter of the telencephalon, medulla, pons, cerebellum, and cord. Astrocytic gliosis was also observed, and under electron microscopy, the vacuoles were seen to be associated with glial cells and to consist of myelin lamellae.42 10

STANDARD

LABORATORY

AND

DIAGNOSTIC

TESTS

The more routine evaluations of electrolytes, hematologic, and renal parameters tend to be unremarkable in these children. Radiologic studies usually do not yield abnormal results. However, some isolated case reports mention ventricular dilation and porencephaly,3 and CT demonstrates hypodensity of white matter and of the corpus callosum. The latter finding may be a specific finding in nonketotic hyperglycinemia.45 In addition to the expected correlates of the paroxysmal activity, the EEG has been repeatedly reported to be consistent with hypsarrhythmic patterns.5, ‘9 32 In the first 2 months of the disease, hypsarrhythmia may be preceded by disorganized polyspikes on the EEG.36 BIOCHEMICAL

FEATURES

With the exception of a single case reported by Perry et a1.,14 significant elevations of glycine in the plasma, urine, and, where determined, in the CSF are a constant feature. As emphasized by Striver, Sprague, and Horwood, the elevated CSF glycine level is of special interest because the ratio of plasma to CSF glycine is abnormally depressed in nonketotic hyperglycinemia. This reduced ratio is not improved when an agent that conjugates and promotes clearance of glzcine is successful in restoring plasma concentrations to normal. Striver and Rosenberg have reported elsewhere45 that the ratio of plasma to CSF concentration of an amino acid tends to be diminished when the metabolism of that amino acid is abnormal in the brain itself. The implication relevant to nonketotic hyperglycinemia is that this disorder includes some specific aberration of glycine metabolism in the CNS. This notion is reinforced by demonstration that glytine-cleavage enzyme activit seems to be depressed in brain tissue from autopsied patients. P4p20925 Since plasma and urine glytine levels are also elevated, the metabolic defect should extend to other tissues, and indeed, the cleavage enzpe activity has been found to be depressed in liver tissue.14’lg. ’ Moreover, Simila and Visakorpi report decreased renal tubular resorption of glycine in a single patient with nonketotic hyperg1ycinemia.s Another indication that the disorder of glycine metabolism may be either diffuse in location or diverse in nature is provided by Baumgartner and co-workers, who demonstrated delayed clearance of labeled glycine from the circulation of patients relative to controls.’ Various workers have.,reported abnormal levels of metabolites other than glycine in the body fluids. The hypo-oxaluria noted in one early case has not been observed subsequently.’ Variable increases in plasma threonine, lysine, histidine, proline, methionine, taurine, isoleucine, and glutamine levels have been re11

ported, while plasma levels of cystine and y-aminobutyric acid have been noted to be decreased. ‘* 4*5*‘* lo* l3 Elevated CSF taurine has also been found.13 However, none of these amino acid increases and decreases has been found consistently. Interesting information relevant to the biochemistry of the disorder has been provided by experiments involving loading of patients with amino acids. Levy first reported that exogenously administered valine caused coma.46 Similarly, Krieger and Hart found that when 15 mg/kg of valine was given to their patient, there was a decrease in spontaneous movements and response to stimuli.47 These results indicate that intermediates in the metabolism of branched chain amino acids affect glycine metabolism.27 The mechanism and significance of the relationships of branched chain amino acids to nonketotic hyperglycinemia remain to be determined. POSSIBLE

ETIOLOGIC

MECHANISMS

How the alteration of amino acid composition in nonketotic hyperglycinemia comes about and then causes neuronal insult remain issues of considerable speculation. Present evidence indicates that this disorder is the manifestation of some primary defect in glycine metabolism.20’ 32*33 As a broad conceptual framework, one can use the delineation of two categories of defects underlying disorders of amino acid metabolism: those involving the deficiency of a specific enzyme or its activity, and those involving abnormal transport systems.48 A constituent gene product of an enzyme complex may be either absent or of such altered structure that assayable enzyme activity is diminished. It is also possible that some essential cofactor is either not available or unable to activate the enzyme complex due to chemical modification of the co-factor molecule or its binding site.48 In phenylketonuria, for example, there may be three distinct metabolic variants. There may be a deficiency of the apoenzyme (phenylalanine hydroxylase) itself, or a deficiency of a necessary co-factor (tetrahydrobiopterin), or the absence of an enzyme needed to regenerate the co-factor (dihydropteridin reductase).4g The presence of some inhibitory substance could also impair enzyme activity and effect changes in glycine metabolism. Hillman and Otto have presented evidence that tiglic acid, an intermediate in isoleucine metabolism, inhibits the interconversion of glycine and serine in cultured fibroblasts.27 THE GLYCINE

CLEAVAGE

ENZYME

The intermediary metabolism of glycine in the normal organism is known to be involved in pathways leading to purine, cre12

atinine, and glutathione formation and, via serine, to pyruvate, ethanolamine, and porphyrin formation. However, most investigators who have studied glycine metabolism in patients with nonketotic hyperglycinemia have concluded that the essential metabolic defect lies in the interconversion of glycine to serine, and vice versa.” 12,14,5o According to Klein and Sagers, this interconversion comprises a two-step reaction sequence in bacterial systems.22 This enzyme system seems to exist in rat liver’ and has also been demonstrated in cell-free systems utilizing human brain and liver,15’ lgl 20*25P28,51 although the constituent protein and co-factors have not been purified in a human tissue preparation. The proposed reaction sequence, with FH4 and FH4 CH20H representing tetrahydrofolic acid and its hydroxymethyl derivative, is as follows: (A) NH2CH2COOH + FH4 + Hz0 + FH4CH20H + NH3 + CO2 (B) FH4CH20H + NH2CH2COOH + HOCH2CHNH2C02 + FH4 Reaction A is the glycine cleavage reaction. In rat brain, it requires nicotinamide-adenine dinucleotide (NAD) and pyridoxal phosphate as co-factors, as well as reducing equivalents.52 The latter were provided as dithiothreitol in Bruin and co-workers’ experimental system,52 but may be available as other sulthydryl compounds in vivo. Reaction B is the serine hydroxymethyl transferase reaction, which is believed to proceed unimpaired in tissues of patients with nonketotic hyperglycinemia.28 The glycine cleavage reaction has been shown to be deficient in liver and brain tissue homogenates from nonketotic hyperglycinemia patients.15’ 20,25 In addition, Nyhan and co-workers’,’ have performed in vivo studies of @4ne in affected patients using labeled amino acids: Either l- C glycine or 2-14C glycine was injected intravenously and labeled CO2 was measured in expired air. In control subjects, the formation of CO2 from 1-14C glycine was rapid, presumably because of an intact glycine cleavage system, while the conversion of 2-14C glycine to labeled CO2 was much slower, since conversion to serine and oxidation in the Krebs cycle via pyruvate are necessary. In patients with nonketotic hyperglycinemia, the rate of conversion of labeled glycine to labeled CO2 was comparably slow, regardless of whether the label was in the C-l or C-2 position. This suggested to Nyhan et al. a deficient glycine cleavage enzyme activity, requiring more extensive metabolism via serine and pyruvate in order for the first carbon to appear as C02.1P7 One additional aspect of the glycine cleavage reaction is that it augments the 1-C pool because of its generation of hydroxymethyl tetrahydrofolic acid. This feature was emphasized by DeGroot et al., who speculated that the reaction might have a central role in the generation of 1-C units necessary for the sur13

viva1 of the organism.” These workers proposed that the increased glycine in nonketotic hyperglycinemia is the result of augmented conversion of serine to glycine owing to 1-C depletion and failure of the cleavage reaction, which normally would ameliorate the 1-C depletion. Tada and co-workersz8 reported that glycine cleavage enzyme activity was deficient in liver homogenates from two patients with nonketotic hyperglycinemia as well as in one patient with ketotic hyperglycinemia. Serine hydroxymethyl transferase activity was normal in all three patients. These findings suggest that deficient cleavage enzyme activity may be a common mechanism in the two disorders. Although the exact nature of glycine cleavage enzyme impairment is unknown, some provocative experimental data have been reported suggesting analogies with the more full2 deMoscribed cleavage enz me system in rat liver mitochondria. tokawa and Kikuchi x 3954 have described an enzyme system consisting of four protein subunits, H, P, T, and L, that occupies the inner membrane of rat liver mitochondria. The activity of this system depends on the presence of NADH as an electron donor, with dihydrothreitol also able to serve this function. The H protein is described as a heat-stable polypeptide with a molecular weight of 17,000 that exists as a functional disulfide molecule and probably contains lipoic acid. The P protein is described as a very unstable protein that uses pyridoxal phosphate as co-factor. The T protein is a relatively stable molecule with a molecular weight of 33,000 that requires the presence of both tetrahydrofolic acid and sulfhydryl groups for activity. The L protein exhibits lipoamide dehydrogenase activity and is described as participating in electron transport between glycine and NADH.539 54 The proposed reaction sequence is as follows: (0 NH&H&OOH (D) @-palp

‘=ECH

=

NCHzCOOH

2 NH 2

SH 0% 0’ ‘S-CH2NH2 SH +@3’ ‘SH 14

+ @-palp

+

G @-palp +

B’s ‘S

= NCH&OOH e

@-palp

+

co 2

0 + H4 folate e NH3 + 5,lO CH2Hk folate

SH +NADH+H+ ‘ S ‘SH In these reactions P, H, T, and L represent the four constituent proteins, palp is pyridoxal phosphate, and THF is tetrahydrofolic acid. Informative data on the activity of the glycine cleavage enzyme in nonketotic hyperglycinemic patients has been provided by an ing;nious series of experiments executed by Perry and cousing the components of a system comparable to the workers, system of Motokawa and Kikuchi. One aspect of their investigations was determination of both glycine content and glycine cleavage activity (as 14COz.evolved from 1-14C glycine) in patients and in control subjects. A twofold to eightfold increase of glycine was found in all nonketotic hyperglycinemia brain areas assayed as compared with extracts from normal brain and from the brain of an infant with hyperglycinemia secondary to methylmalonic acidemia. There was also complete absence of glycine cleavage enzyme activity in the nonketotic hyperglycinemia brain. In a second series of experiments, homogenized brain tissue from nonketotic hyperglycinemia patients was dialyzed against an isotonic solution of sucrose and dihydrothreitol, and cleavage enzyme activity was determined before dialysis and 24 and 48 hours after dialysis. In control brain there was a gradual increase in enzyme activity over time. This contrasted markedly to the situation in nonketotic hyperglycinemia brain, in which enzyme activity remained undetectable despite prolonged dialysis. A final component of Perry’s investigations involved an assay of cleavage enzyme activity after recombination of brain homogenates from both patients and controls with partially purified enzyme protein fractions from the bacterium A. globoformis. Two fractions, one containing the P, T, and L proteins and one containing the H protein, are separable from this bacterial system based on solubility characteristics. It was found that control brain homogenate activated the isolated P, T, and L fraction, while no glycine cleavage enzyme activity resulted from addition of nonketotic hyperglycinemia brain homogenate to the same fraction. However, neither control nor nonketotic hyperglycinemia brain activated the separated H fraction, presumably because of the instability of the mammalian P protein.259 53954 These recombination experiments indicate that deficient activity of the glycine cleavage enzyme system in infants with. nonketotic hyperglycinemia results at least in part from impairment of H protein function, since control and nonketotic hyperglycinemia brain homogenates both failed to activate the H protein fraction. Of particular interest was the finding that with 03 0’

+NAD%&

15

prolonged dialysis there was an increase in assayable enzyme activity in control homogenates that was totally lacking in the nonketotic hyperglycinemia homogenates. Perhaps this phenomenon reflects the gradual removal of some enzyme inhibitor that in nonketotic hyperglycinemia patients, for unknown reasons, resists removal of its effects on the enzyme, as was seen when normal brain samples were added. Also, Daly and co-workers found that homogenates from several regions of rat CNS demonstrated a 25-fold range in enzyme activities, and that activities were not additive when homogenates from various areas were combined.55 The conclusion that the brain contains an endogenous inhibitor of glycine cleavage enzyme activity with varying activity in different CNS regions is further supported by the finding by Daly et al. that CNS homogenates inhibit glytine cleavage activity of rat liver homogenates.K5 It is possible, therefore, that the suppression of glycine catabolism via the cleavage reaction in patients with nonketotic hyperglycinemia may result from the presence of an unusually resilient activity of some uncharacterized enzyme inhibitor. This inhibitor may be an intermediary metabolite lacking an obvious relationship to glycine biochemistry. For example, Hillman and Otto” have demonstrated that tiglic acid, a product of isoleucine catabolism, is capable of inhibiting the glycine cleavage reaction. The hypothesis that some unknown primary metabolic defect causes alterations in subcellular concentrations of intermediate products, which in turn cause inhibition of activity of the cleavage reactions involving the H protein, is an attractive alternative to the hypothesis that nonketotic hyperglycinemia is caused primarily by a structurally abnormal apoenzyme. However, a recent series of elegant experiments by Hiraga et a1.20has yielded further evidence of the nature of H protein dysfunction. The content of H protein in the liver of a nonketotic hyperglycinemic patient was 35% that of control human liver, while specific activity of the patient’s H protein was only 4% that of control H protein. In addition, unlike control H protein, the H protein of the patient did not react with lipoamide dehydrogenase, and titration of thiol groups suggested that the patient’s H protein lacked lipoic acid. The absence of lipoic acid in the H protein was concluded to be the primary molecular lesion in the disorder,20 which implies that the apoenzyme may indeed be structurally abnormal. Thus, available data on the glycine cleavage enzyme from both in vivo and in vitro experiments have documented its diminished activity in nonketotic hyperglycinemia patients. The decrease in activity has been localized to a function corresponding to the H protein in the chemical sequence outlined previously. The enzyme inhibition may be a consequence of the presence of some as yet unidentified metabolite accumulating from 16

a primary defect in some uncharacterized separate pathway. The argument against deficient co-factor availability as the primary biochemical abnormality is based on lack of consistent improvement when co-factor loading is used as a therapeutic approach (Table 2). Structurally abnormal glycine cleavage enzyme has not been excluded as a concomitant or causal factor in nonketotic hyperglycinemia; however, and the apparent lack of lipoic acid in the H protein may reflect abnormal structure of the apoenzyme.20 Further studies will be necessary to determine the exact mechanism of decreased glycine cleavage activity in the disease. APPARENT ASSOCIATION CoA CARBOXYLASE

OF DEFICIENT

PROPIONYL

Multiple biochemical pathways may be involved in nonketotic hyperglycinemia, and indeed, a second enzyme system has been implicated as contributing to the metabolic abnormality. This TABLE

P.-ATTEMPTED

THERAPEUTICAPPROACHESAND THEIR RESULTS IN INFANTS WITH NONKETOTIC HYPERGLYCINEMIA

TREATMENT Protein restriction

None Improvement Initial improvement None None Improvement None Improvement Unknown

Plasma glycine normal None None None None None Plasma glycine decreased Plasma and CSF glycine decreased CSF glycine increased

11 12 12,14,* 77

None None None None None None None

Plasma glycine decreased None None None Plasma glycine decreased None None

17: 79*

Improvement

77*

Improvement

Plasma and CSF glycine decreased None

6,7* 13,* 79* 16*

Ventriculoperitoneal shunt Augmentation of 1-C pool with: Formate Formate Leucovorin Methionine Methionine Choline Co-factor supplementation (app and folic

*Combined treatment

None

10,* 13* 12,14*

Conjugation of glycine with benzoate or salicylate

BIOCHEMICAL RESPONSE

None

4,10,* 12,14 599 7* 7*

Exchange transfusion

Glycine receptor antagonist (strychnine)

CLINICAL RESPONSE

REFERENCE

16* 12 14,* 77* 12,76 12

modalities were used. 17

enzyme is propionyl CoA carboxylase, which utilizes CO2 and adenosine triphosphate (ATP) and converts propionyl CoA to Dmethylmalonyl CoA during either fatty acid oxidation or isoleutine metabolism as follows: (G) Propionyl CoA + ATP + COz e ADP + P + D-methylmalonyl CoA (H) D-methylmalonyl CoA Z$ L-methylmalonyl CoA (I) L-methylmalonyl CoA = succinyl CoA Farriaux and co-workers have described a patient in whom hyperglycinemia was associated with absent glycine cleavage activity and reduced propionyl CoA carboxylase activity.50 In addition, Revsin et al. 56 demonstrated significant reduction of propionyl CoA carboxylase activity in fibroblasts derived from a patient whom they described as having nonketotic hyperglycinemia. This reduction was found to be improved by addition of valine to the media. These reports of deficient propionyl CoA carboxylase activity imply that the diagnosis of nonketotic hyperglycinemia can be made by demonstrating deficient glycine cleavage enzyme activity. We are inclined to disagree with this assertion because the metabolic pathway outlined in reactions G, H, and I includes enzymes known to be involved in the ketotic hyperglycinemia syndromes.2g-31 Since depression of glycine cleavage activity has been recognized in ketotic hyperglycinemia,28 and since the catabolic products of branched chain amino acids may affect inhibition of this enzyme in vitro,27 we suggest that demonstration of such inhibition, while perhaps a necessary biochemical correlate of the syndrome, is not in itself sufficient to establish the diagnosis of nonketotic hyperglycinemia. As will be emphasized in the final section of this monograph our preference, is to rely on a characteristic pattern of elevated glycine concentrations in central spinal fluid, blood, and urine associated with a distinct clinical presentation. ROLE OF GLYCINE

TRANSPORT

The second general category of amino acid disorders, disorders of impaired transport mechanisms, has been described by Scriber and Rosenberg.45 These authors make several observations pertinent to nonketotic hyperglycinemia. It is apparent that a transport disorder may actually be a specific form of enzyme deficiency, since amino acid transport seems to depend on cellular energy metabolism and to be mediated by genetically encoded membrane proteins. It is clear that induction, repression, and feedback inhibition may influence transport processes in the same way that they affect intracellular enzymes, and that transport mechanisms seem to be co-factor dependent.46 18

Several researchers have investigated glycine transport in the CNS. Murray and Cutler conducted ventriculocisternal and ventriculolumbar perfusion experiments, administering labeled glytine and inulin to living cats.67P58 The results of their studies suggested first-order, saturable kinetics for transport of glycine from the lateral ventricle to the brain parenchyma, suggesting a carrier-mediated, relatively specific system that was not inhibited by leucine, alanine, lysine, or proline. Their findings were interpreted as demonstrating two transport sites, the choroid plexus and the spinal subarachnoid space. In a subsequent study, Murray administered labeled glycine to rats via the carotid artery and then collected venous samples from the confluens of sinuses.5g In this experimental preparation, labeled glycine uptake was decreased in the presence of unlabeled glycine. There was also inhibition by phenylalanine and proline, and this inhibition was found to be stereospecific for L amino acids. Murray concluded that a carrier-mediated glytine transport was present at the blood-brain interface. However, further experiments by others5g-62 have provided conflicting data on carrier-mediated glycine transport in the CNS. Work on rat brain by Valdez et al. suggests that a high-affinity transport system for glycine may be present in myelin and in some glial plasma membranes, but not in the nuclei, mitochondria, endoplasmic reticulum, or synaptosomes.63 These limited data on CNS transport of glycine indicate the potential for impaired carrier-mediated transport at two CNS sites, the interface between CSF and brain and between capillary blood and brain. While there have been no reported investigations of the specific phenomenon of glycine transport in the nervous systems of patients with nonketotic hyperglycinemia, comparisons have been made between transport in nonketotic hyperglycinemia fibroblast lines and human diploid fibroblast controls. Revsin and Mori-0~~~ presented data indicating that glycine is transported at a reduced rate and that the intracellular content of glycine in fibroblasts of patients with nonketotic hyperglycinemia is less than that of controls. Transport in both cell lines was found to require metabolic energy, sulfhydryl groups, sodium, and potassium and to exhibit cross-inhibition by proline, valine, and alanine. Kinetic analyses in these experiments revealed that the control and patient cell lines had a similar K, but that the patient cell lines had a lower Vmax.64 However, more recent work by Kelly et a1.23has failed to support the results and conclusions of Revsin and Morrow.64 Kelly et al. measured glycine uptake into nonketotic hyperglycinemia cells at l-minute rates, whereas Revsin and Morrow measured transport over 20 minutes. Kelly and co-workers also measured 19

the extent of glycine incorporation into protein and the oxidation of glycine and found no differences between patients with nonketotic hyperglycinemia and control subjects. In their experiments, there was no evidence of cross-inhibition, and the K, and V,, values for each group overlapped. Hence, nonketotic hyperglycinemia and normal cells could not be reliably distinguished on the basis of glycine transport, incorporation into protein, or oxidation. This contradiction to the earlier results of Revsin and Morrow is thought by Kelly and his co-investigators to be due either to some difference in the control fibroblast lines used by the two groups or perhaps to genetic heterogeneity in either the nonketotic hyperglycinemia or control cell lines. Subsequent studies by Halton and Krieger65 also failed to demonstrate a difference in glycine transport between patient and control lines with respect to K,, V,, or Ki. At present, the existence of a specific derangement in glycine transport in nonketotic hyperglycinemia has not been established with certainty, although further investigations in this area would be useful. The question of transport of the amino acid between intracellular compartments, especially into mitochondria, would be of interest, since a process of diminished induction conceivably could explain reduced activity of enzymes using glycine as substrate. As noted previously, available evidence suggests that more than one enzyme deficiency may accompany this metabolic disorder. POSSIBLE

MECHANISMS

OF HYPERGLYCINEMIA

The biochemical changes that mediate the observed hyperglycinemia have not been fully elucidated. Diminished flux through a major catabolic pathway could adequately explain the hyperglycinemia, and the decreased glycine cleavage activity might contribute to high glycine levels, although the activity of this reaction relative to other reactions in human cells that utilize glycine is unknown. The possibility that propionyl CoA carboxylase inhibition might be an associated phenomenon in nonketotic hyperglycinemia suggests a second site where glycine utilization might be diminished: Stumpf and co-workers have demonstrated a direct inhibition of succinyl CoA ligase by propionyl CoA in progm-iic acidemia, one of the ketotic hyperglycinemia syndromes. 9 Succinyl CoA ligase catalyzes the interconversion of succinate and succinyl CoA, and one might hypothesize that its inhibition could diminish utilization of glycine via the S-aminolevulinic acid synthetase reaction:

20

Succinate

Succinyl CoA ligase JO ’ ’ ’ ’ _ ’ . _ . , Succinyl CoA + glycine i TO a-Amino-(5ketoadipic acid Propionyl CoA 4 -+coz b-Aminolevulinic acid

J

Heme synthesis While it is not clear that the succinyl CoA utilized in this reaction is formed directly from succinate, there is some experimental evidence that acyl CoA derivatives may inhibit a proposed succinate-glycine cycle.67, 68 At the least, these hypotheses demonstrate how intracellular enzyme deficiencies may result in abnormal levels of substrates which superficially lack a clear relationship to the known chemical lesion. Disorders known to involve mitochondrial enzymes in particular provide further illustration of how incompletely understood inhibitory processes may cause a circumscribed enzyme abnormality to be associated with broader impairment of biochemical processes. One example is the common finding of hyperammonemia in patients with primary disorders of organic acid and branched chain amino acid metabolism, in which the deficient enzymes are zs-gparently a distinct from the enzymes mediating ureagenesis. 133 Interestingly, recent evidence indicates that here again, acyl CoA derivatives function as agents of inhibition, causing diminished activity of urea cycle enzymes.6g Increased circulatory glycine concentration in nonketotic hyperglycinemia might represent such a diffuse state of chemical inhibition resulting from an unknown primary event. However, if this is the case, the disproportionate elevation of CSF glycine relative to plasma and urine levels in nonketotic hyperglycinemia suggests a primary chemical defect with its fullest expression in the CNS. Mechanisms other than diminished utilization of glycine might be responsible for elevation of glycine levels in body fluids. Overproduction of either glycine itself or of some substrate readily convertible to it would cause such elevations, as would impaired transport between intracellular compartments. To date no experimental work has implicated these mechanisms as causing hyperglycinemia, and the kind of utilization inhibition described in the preceding paragraph may be a promising object of investigation. PATHOGENESIS

OF NEUROLOGIC

DEFICITS

Although glycine is known to be elevated in the plasma, urine, and CSF of patients with nonketotic hyperglycinemia, it 21

is not well understood how these elevations translate into the severe associated neurologic impairments. Significant evidence has accumulated indicating glycine to be an inhibitory postsynaptic receptor in the mammalian spinal cord and brain stern.” Snyder and his associates have succeeded in studying glycine rece tors with radiolabeled strychnine, a potent glycine antagonist sfl, ‘l Further work by this group revealed that the binding of 3h-strychnine to the glycine receptor was specifically inhibited by the benzodiazepine compounds51 and that the affinity of these drugs for the glycine receptor correlated well with their potency in behavioral tests on animals. These results were interpreted by Snyder to mean that benzodiazepines exert their pharmacologic action by stimulating the central postsynaptic glycine receptor and that strychnine is inhibitory to this receptor. The role of benzodiazepine in this system, however, is controversial. Curtis and co-investigators presented evidence that the inhibitory effect of strychnine on the action of glycine in cat dorsal root inter-neurons was not reduced by intravenous diazepam administration.72 The pharmacologic properties of the central glycine receptor system were invoked by Ransom to explain the pathophysiology of the neurologic abnormalities associated with the nonketotic hyperglycinemia syndrome.73 This investigator found that dissociated cultures of mouse spinal cord displayed inhibitory responses to glycine. When the cell cultures were studied in the presence of glycine, they became insensitive to the effects of further glycine application; chronic glycine treatment also produced desensitization to glycine inhibition.73 This diminished sensitivity to the normal inhibitory effects of glycine could explain how its excessive levels in the extracellular fluids bathing the nervous systems of affected patients might cause hjperexcitability, seizures, and increased muscle tone. This hypothesis can be carried one step further, since desensitization may lead to an increase in receptor number or sensitivity, a phenomenon of apparently wide physiologic importance in the CNS.74 Perhaps the variations in muscle tone and the lethargy and seizures in these patients can be related to a fluctuating balance between desensitization and rebound hypersensitivity of the central glycine receptor. This hypothesis is consistent with the known physiology of the glycine receptor system as well as with the usual clinical observations. THERAPEUTIC

CONSIDERATIONS

Earliest attempts to modify the poor prognosis of patients with nonketotic hyperglycinemia aimed at decreasing glycine levels in the extracellular fluid. Three types of therapeutic approaches have been instituted to effect this decrease: dietary protein re22

striction, exchange transfusion, and promotion of renal clearance by chemical conjugation (see Table 2). Several investigators were able to depress plasma glycine levels to normal by restricting protein intake to 1 gm/kg/day or less; however, there was no concomitant clinical improvement.5Pg Other reports mentioned that there were no positive clinical or biochemical .changes after protein restriction4* lo912*‘* When Baumgartner and co-workers gave their patient a low protein diet followed by exchange transfusions on days 7 and 8 of life, they observed an initial improvement in feeding and social responsiveness as well as a decrease in seizure activity. This child subsequently deteriorated neurologically.’ Similarly, attempts to diminish glycine load by exchange transfusion did not lead to sustained clinical progress in the patients of Okken and collaborators” and Bachmann and co-investigators.13 This modality of treatment also failed when combined with other agents.38 There have been numerous efforts to promote condu$ation of glycine with administered salicylate or benzoate.69 ’1 14,“* l7 Glycine combines with these agents to form salicuric and hippuric acids, respectively. Ziter et al. noted that a patient given sodium benzoate and aspirin became more alert and less irritable, but that seizures, abnormal reflexes, and severe developmental delay continued.6 Of interest is one of the patients discussed by Krieger et al., who experienced improvement in breathing and feeding behavior while receiving sodium benzoate.16 At the same time, the plasma and CSF glycine levels were noted to be decreased. When the benzoate was discontinued and later reinstituted, these authors noted a pattern of deterioration and improvement, respectively, which corresponded with fluctuation of glycine levels in the CSF. While these results are encouraging, it must be stressed that clinical recovery has not occurred in several other patients who received trials of sodium benzoate.12-l4 A novel treatment approach was introduced by Krieger et a1.,16who attempted to effect a “washout” of glycine from the brain via a ventriculoperitoneal shunt. Although there was no resulting change in glycine concentration in the ventricular fluid, an increase in lumbar CSF glycine was demonstrable. The authors speculated that a net flux of glycine from the brain’s extracellular space to the CSF may have been caused by the shunt.16 The realization that the clinical syndrome of nonketotic hyperglycinemia is accompanied by altered intracellular enzyme activity led to efforts at amelioration by co-factor supplementation. DeGroot and his colleagues argued that deficient glycine cleavage activity causes depletion of the 1-C units necessary for normal metabolism.” They thought that hyperglycinemia might 23

in turn result from a compensatory acceleration of conversion of serine to glycine in response to the 1-C depletion. These authors provided 1-C units such as methionine and found subsequently normal glycine levels despite clinical deterioration.” Trijbels and co-workers extended this investigation of co-factor supplementation to several 1-C donors, including leucovorin, methionine, choline, and formate, as well as using pyridoxine and folic acid. Formate was the only compound that decreased the lasma glycine level, but no clinical improvement was observed. fi Similarly, Speilberg and collaborators did not find leucovorin to be of benefit.75 Administration of pzridoxine and folic acid analogues by both Perry et al. and Gitzelman and coinvestigators 76 failed to cause either clinical or biochemical changes in their patients. The refractory seizures that occur uniformly as part of the disorder have been treated with combinations of benzodiazepines, phenytoin, ACTH, and valproic acid. There is evidence that valproate may be especially useful in this capacity, despite reports that it occasionally elevates urine and plasma g;Tcine levels in patients not having nonketotic hyperglycinemia. Evidence implicating strychnine as an inhibitor of glycine receptors in the CNS has motivated several investigators to use strychnine as a therapeutic agent. Gitzelman and his group76 began their patients on a dose of 0.4 mgkg/day but then tapered the dose to 0.3 mg/kg/day because of increased seizure activity. They observed improvement of muscle tone, motor control, and attentiveness, as well as a social smile and reaction to pleasurable stimuli. These effects deteriorated when strychnine was discontinued but improvement recommenced when therapy was resumed.76 This patient was also receiving clonazepam, a benzodiazepine anticonvulsant. Positive results with strychnine therapy were also achieved by Arneson and colleagues, who observed increased alertness, decreased seizure activity, improvement in vomiting, and less involuntary movements when their patient was given 0.1 mg/kg/day of st chnine in conjunction with clonazepam and sodium benzoate.%i In a trial of strychnine (0.2-0.9 mg/kg) in three Finnish children in whom the disorder was diagnosed in the first hours of life, death occurred before age 10 days, despite the concomitant use of exchange transfusions.38 Hence, although there is some indication that strychnine might lessen the degree of neuromotor impairment, there is also evidence that its use does not halt progression to death in severe cases. Even with this therapeutic approach, the degree of psychomotor retardation might well be significant, and the usefulness of strychnine is limited by the danger of toxicity. We combined several of the above treatment modalities in the management of twins with nonketotic hyperglycinemia. Phar24

macologic doses of pyridoxine and nicotinamide were given along with leucovorin, sodium formate, benzoate, sodium salicylate, clonazepam, and strychnine in various combinations in an effort to achieve positive synergistic effects. Unfortunately, the patients died before any meaningful conclusions could be drawn. The combination of strychnine and sodium benzoate has been found to be of some use in slowing the disease’s progression.78 Further attempts to combine therapeutic strategies that singly fail to have a significant influence on the clinical course of the disease should be encouraged.7g PROPOSED DIAGNOSTIC HYPERGLYCINEMIA

CRITERIA

FOR NONKETOTIC

It is not surprising that a disorder of such incompletely understood causality is difficult to define with precision. Our clinical studies and our review of the literature on nonketotic hyperglycinemia have convinced us that it is a discrete diagnostic entity with features that distinguish it from other inborn metabolic derangements with which it is likely to be confused. Disorders with features similar to nonketotic hyperglycinemia include pketothiolase deficiency, propionyl CoA carboxylase deficiency, the methylmalonic acidemias, and carbamyl phosphate synthetase deficiency. Their distinction from nonketotic hyperglycinemia on clinical grounds would be of practical usefulness, since all of these syndromes may be associated with hyperglycinemia and hyperglycinuria yet have significantly better prognoses than nonketotic hyperglycinemia. Because some details of the pathogenesis of nonketotic hyperglycinemia remain unknown, the following criteria necessarily are descriptive criteria that apply to a group of patients with common clinical and chemical features of disease. The emerging definition will be a restricted one: It will delineate a group of children with a degenerative disorder beginning quite early in life and progressing inexorabp to severe debility or death. Atypical and late-onset variants3 735 will therefore be excluded, although at least one instance has been reported in which the onset of clinical features very similar to those of the typical earlyonset syndrome occurred after six months of normal development.” This exclusion seems appropriate until a specific etiologic biochemical defect can be uncovered. Lacking the basic knowledge that would facilitate broad application of the term “nonketotic hyperglycinemia,” we prefer to apply it only to those patients fulfilling the following criteria: 1. Onset during the first 72 hours of life, with early munifestations of seizures, reduced level of consciousness, poor feeding, and apnea. In the affected infants, these findings will not be

readily explicable on the basis of infection, trauma, hypoxia, ab25

errations of glucose, calcium, bilirubin, and electrolyte levels or other commonly encountered problems in infants. 2, Absence of ketoacidosis, as indicated by early determination of plasma bicarbonate levels, arterial or capillary blood pH, and either serum or urine ketones. These investigations are needed to exclude organic acidemias. 3. Elevation of glycine levels in plasma, urine, and CSF, with clearly disproportionate elevation of CSF levels. The ratio of plasma to CSF glycine concentration is usually less than 5 in nonketotic hyperglycinemia patients, while in normals and in patients with other forms of hyperglycinemia it is 30 or greater.44 4. Relentlessly progressive mental retardation, developmental delay, and spasticity, and emergence of an EEG pattern indicating significant diffuse abnormalities, including hypsarrhythmia. These features certainly will be evident during the first year of life but may manifest in the first weeks.36 5. Negative findings supporting the diagnosis include lack of response to protein restriction, lack of recurrent vomiting, and absence of neutropenia, thrombocytopenia, and osteoporosis. In practice, any infant with the suggestive clinical and laboratory findings outlined in the first two criteria should have amino acid levels determined. Should hyperglycinemia be determined by a urine amino acid screen, it is essential to obtain quantitative glycine levels on plasma and CSF. Once the disproportionate CSF elevation (third criterion) is established, the presumptive diagnosis of nonketotic hyperglycinemia may be made. This should be possible during the first week of life in most centers. The final two criteria bear out the early diagnosis over the subsequent weeks and months. Quantitative determination of organic acid levels and specific enzymatic studies may be useful at a later time. However, we are suggesting that an early diagnosis is possible by observing progression of the initial symptoms and by the use of a limited number of laboratory investigations. SUMMARY

The salient features of nonketotic hyperglycinemia include apnea, feeding difficulties, lethargy, seizures, abnormal muscle tone and reflex activity, significant developmental delay, and, in most instances, early death. The pathogenesis of the biochemical defect leading to increased glycine concentration in blood, urine, and CSF is likely to concern derangements of the glycine cleavage enzyme and/or transport mechanisms of glycine. Our current state of knowledge of this disorder is incomplete. Therapeutic attempts, as described in Table 2, have been largely unsuccessful. 26

Further basic research on the underlying biochemical perturbation, including additional documentation of the glycine cleavage enzyme deficiency patterns, of substrate inhibition of key metabolic pathways, and of glycine transport aberrations, as well as investigations of new pharmacologic approaches, will be a challenge for investigators in this field. It is hoped that new knowledge in these areas will eventually lead to reduction of morbidity and mortality in children with nonketotic hyperglycinemia. ACKNOWLEDGMENTS

Dr. Richard E. Hillman reviewed the manuscript and provided much appreciated editorial advice. Robin O’Reilly provided outstanding secretarial assistance. REFERENCES 1. Nyhan W.L.: Nonketotic hyperglycinemia, in Stanbury J.B., Wyngaarden J.B.. Frederickson D.S. (eds). The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1978, pp. 518-527. 2. Mabry C.C., Karam E.A.: Idiopathic hyperglycinemia and hyperglycinuria. South. Med. J. 56:1444, 1963. 3. Gerritaen T., Kaveggia F., Waisman H.A.: A new type of hyperglycinemia with hypo-oxaluria. Pediatrics 36:882, 1965. 4. Balfe J.W., Levison H., Hanley W.B., et al.: Hyperglycinemia and hyperglycinuria in a newborn. Can. Med. Assoc. J. 92:346, 1956. 5. Rampin S., V&her O., Curtius H.C., et al.: Hereditaire hyperglycinamie. Helv. Paediutr. Acta 22:135, 1967. 6. Ziter F.A., Bray P.F., Madsen J.A., et al.: The clinical findings in a patient with nonketotic hyperglycinemia. Pedintr. Res. 2:250, 1968. 7. Baumgartner R., Ando T., Nyhan W.L.: Nonketotic hyperglycinemia. J. Pediatr. 751022, 1969. 8. Simila S., Visakorpi S.K.: Clinical findings in three patients with nonketotic hvnerelvcinemia. Ann. Clin. Res. 2:X1-156, 1970. 9. FkEdiiaid W., Gordon R.R., Owen G.: Nonketotic hyperglycinemia: Clinical findings and amino acid analyses on the plasma of a new case. Clin. Chim. Acta 30:745, 1970. 10. Okken A., DeGroot C.S., Hommes F.A.: Nonketotic hyperglycinemia. J. Ped&r. 77:164, 1970. 11. DeGroot C.J., Trolelstra J.A., Hommes F.A.: Nonketotic hyperglycinemia: An in vivo study of the glycine-serine conversion in liver of three patients and the effect of dietary &hionine. Pediatr. Res. 4:238, 1970. 12. Triibels J.M.F.. Monnes L.A.H.. van der Zee S.P.M.. et al.: A Datient with no&etotic htierglycinemia: Biochemical findings’ and therapeutic approaches. Pediatr. Res. 8:598, 1974. 13. Bachmann C., Mikatsch M.J., Baumgartner R.E., et al.: Nicht-ketoische hyperlgyziniimie. Helv. Paediatr. Acta 26:288, 1971. 14. Perry T.L., Urquhart N., Maclean J., et al.: Nonketotic hyperglycinemia:. Glycine accumulation due to lack of glycine cleavage in brain. N. Engl. J. Med. 292:1269, 1975. 15. Gitzelmann R., Steinmann B., Otten A., et al.: Nonketotic hyperglycinemia treated with strychnine, a glycine receptor antagonist. Helv. Paediatr. Acta 32:517, 1977. 16. Krieger I., Winbaum E.F., Eisenbray A.B.: Cerebrospinal fluid glycine in 27

nonketotic hyperglycinemia: Effect of treatment with sodium bensoate and a ventficulm &unt. Metabolism 26517, 1977. 17. Warburton D., Keats J., Boyle R., et al.: Nonketotic hyperglycinemia: Effects of strychnine therapy. Am. J. Dis. Child. 134:273, 1980. 18. Trauner D.A., Page T., et al.: Progressive neurodegenerative disorder in a patient with nonketotic hyperglycinemia. J. Pediutr. 98:272, 1981. 19. Tadashi Y., Kikuchi Tada K., et al.: Physiologic significance of a glycine cleavage system in human liver as revealed by study of a case of hyperglycinemia. Biachim. Btiphys. Actu 35577, 1969. 20. Hiraga K., Kochi H., Hayasaka K., et al.: Defective glycine cleavage system in nonketotic hyperglycinemia. J. Clin. Invest. 68:525, 1981. 21. Newman E.G., Magasonik B.: The relationship of serine-glycine metabolism to the formation of single carbon unite. Biochim. Biophys. Actu 78:437, 1963. 22. Klein S.M., Sagers R.D.: Glycine metabolism: I. Properties of the system catalyzing the exchange of bicarbonate with the carboxyl group of glycine in peptococcus glycinophilus. J. Biol. Chem. 241:197, 1966. 23. Kelley J.C., Otto E.F., Hillman R.E.: Glycine transport by human diploid fibroblasts: Absence of a defect in cells from patients with nonketotic hyperglycinemia. Pediutr. Res. 13:127-130, 1979. 24. Kikuchi G.: The glycine cleavage system: Composition, reaction and physiological significance. Mol. Cell Biochem. 1:169, 1963. 25. Perry T.L., Urquhart N., Hansen J.: Studies of the glycine cleavage system in brain from infants with glycine encephalopathy. Pediutr. Res. X2:11921197,1977. 26. Hillman R.E.: Personal communication. 27. Hillman R.E., Otto L.F.: Inhibition of glycine-serine interconversion by products of isoleucine metabolism. Pediutr. Res. 8941, 1974. 28. Tada K., Corbeel L.M., Eeckles R., et al.: A block in glycine cleavage reaction as a common mechanism in ketotic and nonketotic hyperglycinemia. Pediatr. Res. 8~721, 1974. 29. Hsia Y.E., Scully K.J., Rosenberg L.E.: Defective propionate carboxylation in ketotic hyperglycinemia. Luncet 1:787, 1969. 30. Morrow G., Barness L.A., Averbach V.H., et al.: Observations of the coexistence of methylmalonic acidemia and glycinemia. J. Pediutr. 74:680, 1969. 31. Ando T., Klingberg W.G., Ward A.N., et al.: Isovaleric acidemia presenting with altered metabolism of glycine. Pediutr. Res. 5:478, 1971. 32. Freeman J.M., Nicholson J.F., Schimke R.T., et al.: Congenital hyperammonemia: Association with hyperglycinemia and decreased levels of carbamy1 phosphate synthetase. Arch. Neural. 23:430, 1970. 33. Hillman R.E., Keating J.P.: Beta-ketothiolase deficiency as a cause of ketotic hyperglycinemia syndrome. Pediatrics 53:221, 1974. 34. Bank W.S., Morrow G.: A familial spinal cord disorder with hyperglycinemia. Arch. Neural. 27:136, 1972. 35. Steinmann G.H., Yudkoff M., Berman P.H., et al.: Late onset nonketotic hyperglycinemia and spinocerebellar degeneration. J. Pediutr. 94:907-911, 1979. 36. Dalla Bernardina B., Aicardi J., et al.: Glycine encephalopathy. Neuropaediutrie l&209, 1979. 37. Garcia-Castro, J.M., Isales-Forsythe, C.M., Levy H.L., et al.: Prenatal diagnosis in nonketotic hyperglycinemia. N. En&. J. Med. 306:79, 1982. . 38. vonWendt L., Seppo S., et al.: Failure of strychnine treatment during the neonatal period in three Finnish children with nonketotic hyperglycinemia. Pediatrics 651166, 1980. 39. vonwendt L., Simila S., et al.: Prenatal brain damage in nonketotic hyperglycinemia. Am. J. Dis. Child. 135:1072, 1982. 40. Shuman R.M., Leech R.W., Scott C.R.: The neuropathology of the nonketotic and ketotic hyperglycinemias: Three cases. Neurology 28:139, 1978. 28

41. Bessman S.P.: Genetic failure of fetal amino acid ‘yustification”: A common basis of many forms of metabolic, nutritional and “nonspecific” mental retardation. J. Pediutr. 811834, 1972. 42. Brun A., Boqjeson M., et al.: Nonketotic hyperglycinemia: A clinical, biochemical and neuropathologic study including electron microscopy findings. Neuropaedintrie 10:195, 1979. 43. Valavanis A., Schuberger O., Hayek J.: Computed tomography in nonketotic hyperglycinemia. Comput. Tomogr. 5:265, 1981. 44. Striver C.R., Sprague W., Horwood S.P.: Plasma-cerebrospinal fluid glycine ratio in normal and nonketotic hyperglycinemia subjects. N. Engl. J. Med. 293:778, 1975. 45. Striver C.R., Rosenberg L.E.: Nature and disorders of amino acid and glytine transport, in Amino Acid Metabolism and Its Disorders. Philadelphia, W.B. Saunders Co., 1973, pp. 178-186. 46. Levy H.L., Nishimura R.N., Erickson A.M., et al.: Hyperglycinemia in in vivo comparison of nonketotic and ketotic (propionic acidemia forms): II. Valine response in nonketotic hyperglycinemia. Pediatr. Res. 16:395, 1972. 47. Krieger I., Hart Z.W.: Valine-sensitive nonketotic hyperglycinemia. J. Ped&r. 85:43, 1974. 48. Swaiman K.F.: Diseases in amino acid metabolism and related conditions, in Swaiman K.F., Wright F.J. (eds.): The Practice of Pediatric Neurology. St. Louis, C.V. Mosby Co., 1975, pp. 359-360. 49. Kaufman S., Berlow S., Summer G.K., et al.: Hyperphenylalaninemia due to a deficiency of biopterin. N. Engl. J. Med. 299:673, 1978. 50. Farriaux J.P., Morel P., Hommes F.A.: Nonketotic hyperglycinemia with increased propionic acid excretion and hyperammonemia. N. Engl. J. Med. 292:588, 1975. 51. Snyder S.H., Enna S.J.: The role of central glycine receptors in the pharmacologic action of benzodiazepines. Adv. Biochem. Psychophurmucol. 14:81, 1975. 52. Bruin W.J., Frantz B.M., Sallach H.J.: The occurrence of a glycine cleavage system in mammalian brain. J. Neurochem. 20:1649, 1973. 53. Motokawa Y., Kikuchi G.: Glycine metabolism by rat liver mitochondria: Reconstitution of the reversible glycine cleavage system with partially purified protein components. B&hem. Biophys. A& 164:624-633,1974. 54. Motokawa Y., Kikuchi G.: Glycine metabolism in rat liver mitochondria: Isolation and some properties of the protein-bound intermediate of the reversible glycine cleavage reaction. B&hem. Biophys. A& 164:634-640, 1974. 55. Daly E.C., Nadi N.J., Aprison M.H.: Regional distribution and properties of the glycine cleavage system within the central nervous system of the rat: Evidence for an endogenous inhibitor during in vitro assay. J. Neurochem. 26:179, 1976. 56. Revsin B., Lebowitz J., Morrow G.: Effect of valine on propionate metabolism in control and hyperglycinemic fibroblasts and rat liver. Pediatr. Res. 11:749, 1977. 57. Murray J.E., Cutler R.W.P.: Carrier-mediated transfer of amino acids from blood to brain. Neurology 23:940, 1973. 58. Murray J.E., Cutler R.W.P.: Clearance of glycine from cat cerebrospinal fluid: Faster clearance from spinal subarachnoid than ventricular compartment. J. Neurochem. 17:703, 1970. 59. Murray J.E.: Transport of glycine from the cerebrospinal fluid. Arch. Neural. 23:23, 1970. 60. Pollay M.: Movement of giycine across the blood-brain barrier of the rabbit. J. Neurobiol. 7:123, 1976. 61. Yudilevich D.V., Sepulveda F.V.: The specificity of amino acid and sugar carriers in the capillaries of dog brain. Adv. Exp. Med. Biol. 69:77, 1976.

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62. DavidoffR.A., Adair R.: GABA and glycine transport in frog central nervous system: High affinity uptake and potassium-evoked release in vitro. Bruin Res. 118:403, 1976. 63. Valdez F., Munoz C., Feria-Velasco A., et al.: Subcellular distribution of rat brain cortex high affinity, sodium dependent transport sites. Brain Res. 122:95, 1977. 64. Revsin B., Morrow G.: Glycine transport in normal and nonketotic hyperglycinemia human diploid fibrobla&. Exp. Cell Res. 100:95, 1976. 65. Halton D.M., Krieger I.: Studies of glycine metabolism and transport in patients with nonketotic hyperglycinemia. Pediatr. Res. 14:932, 1980. 66. Stumpf D.A.: Mitochondrial multisystem disorders: Clinical biochemical and morphological features, in Tyler H.R., Dawson D.M. (eds.): Current Neurology. Boston, Houghton Mifflin Co., 1979, vol. 2. 67. Stumpf D.A., McAfee J., Parks J.K.: Propionate inhibition of succinyl CoA ligase and the citric acid cycle in mitochondria. Pediatr. Res. 14:1127, 1980. 68. Nemeth A.M., Russell C.S.., Shemin D.: The succinate-glycine cycle: II. Metabolism of 6-aminolevulinic acid. J. Biol. Gem. 229:4X, 1957. 69. Gruskey J.A., Rosenberg L.E.: Inhibition of hepatic mitochondrial carbamyl phosphate synthetase by acyl CoA esters: Possible mechanisms of hyperammonemia in the organic acidemias. Pediutr. Res. 13:475, 1979. 70. Iverson L.L., Bloom F.E.: Studies of uptake of 3H-GABA and 3H-glycine in slices and homogenates of rat brain and spinal cord by autoradiography. Bruin Res. 41:131, 1972. 71. Snyder S.H., Young A.B., Bennett J.P., et al.: Synaptic biochemistry of amino acids. Fed. Proc. 32:2039, 1973. 72. Curtis D.R., Game C.J.A., Lodge D.: Benzodiazepines and central glycine receptors. Br. J. Pharmncol. 56~307, 1976. 73. Ransom B.R.: Possible pathophysiology of neurological abnormalities associated with nonketotic hyperglycinemia. N. En&. J. Med. 294:1295-1296, 1976. 74. Snyder S.H.: Receptors, neurotransmitters, and drug responses. N. Engl. J. Med. 300:465, 1979. 75. Speilberg S.P., Lucky A.W., Schulman J.D., et al.: Failure of leucovorin therapy in nonketotic hyperglycinemia. J. Pediutr. 89:631, 1976. 76. Gitzelmann R., Steinmann B., Cuerod M.: Strychnine for the treatment of nonketotic hyperglycinemia. N. Engl. J. Med. 298:1424, 1978. 77. MacDermot K., Nelson W., et al.: Valproate in nonketotic hyperglycinemia. Pediatrics 65:624, 1980. 78. Arneson D., Chien L.T., Chance P., et al.: Strychnine therapy _- in nonketotic hyperglycinemia. Pediubics 63:369, 1979. 79. Pueschel S.M., Boylan J., Langan T.: Therapeutic attempts in infants with nonketotic hyperglycinemia. J. Ment. De@ Res., to be published.

SELF-ASSESSMENT

1. 2. 3. 4. 5.

b,c a,b&e b,c&d b

c

30

ANSWERS

6.a&c 7. c&d 0. a,b&d 9. a, b, c & d 10. a, b, c & e