Brain (1970) 93, 413^*22.

ARGININOSUCCINIC ACIDURIA

CASE REPORT WITH NEUROPATHOLOGICAL FINDINGS

IBY

P. D. LEWIS1 AND A. L. MILLER

(From The National Hospital, Queen Square, London, W.C.I, and The Courtauld Institute ofBiochemistry, Middlesex Hospital Medical School, London, W.I)

ARGINTNOSUCCINIC ACIDURIA, a rare, familial disease with mental and neurologicalfeatures and an abnormality in the Krebs-Henseleit urea cycle, was first recognizedby Allan et al. in 1958. Since then a further 15 cases have been reported {seeBaumgartner et al., 1968; Moore et al., 1968). Studies on the urea cycle enzymes inthe liver of a boy who died of this disorder at the age of 16 were described by Millerand McLean (1967), and the purpose of the present report is to present the clinicaland pathological findings in this patient.

ICASE REPORT

The patient's parents are healthy and not consanguineous. A sister, who was mentally retarded,died at 14 months. No certain diagnosis was made in her case, and no details of her final illnessare available. An extended family history is shown in fig. 1.

9 o* o" 9

"MYXOEDEMA"

D.12 YEARS

MATERNAL GRANDMOTHER S U R V I V E S

C O U S I N H A O U M O N G O L " C H I L D

- I - - - - -I -

1. D-NEPHRITIS"2 KILLED IN WAR

O 6 6 16 20* 6"* * * *

FATHERI 1 I •

O 6*

MOTHER

ALL COUSINS I 1 .A L I V E A N D W E L L 9 . ^ $ ° . 0" 2

PATIENT D 14 MONTHS

6 6 6 6 0 6 0 0 0 6 0 ALL ALIVE AND WELL

FIG. 1.—Family of patient.

—i OTHER AUNTSQ AND UNCLES* ALIVE AND WELL

o o* o" $ o" a*

I9

'Present address: Royal Postgraduate Medical School, Hammersmith Hospital, London, W.12.

24 BRAIN—VOL.

414 P. D. LEWIS AND A. L. MILLER

The patient (M. H. number G 54694) admitted under the care of Dr. Michael Kremer had anormal birth and weighed 7ilb. His early development was slow: he stood at 18 months andwalked at 2 years. When he was 4 he had the first of a number of episodes of unsteadiness anddrowsiness. On this occasion, he was found in a garden shed with a spilt bottle of nicotine next tohim. He was thought to be suffering from nicotine poisoning and was admitted to hospital wheregastric lavage was carried out. Following this, his drowsiness deepened and he sank into a comawhich lasted for twenty-four hours and then lifted spontaneously. In the following year his hearingwas found to be defective, and he later attended a school for partially deaf children. About thistime his parents noted his hair to be unusually fine. At 8 he had another attack of drowsiness, thistime with convulsive jerking of the limbs followed by unconsciousness for forty-five minutes.Further attacks occurred when he was 13, some lasting for three days. He continued to attendschool, though he was thought to be three to four years behind his classmates.

Two months before his final admission to hospital he was found asleep amidst noisy children.He was roused with difficulty, but remained drowsy and very unsteady after this, with slurred speechand impaired manual ability. Five days before admission his consciousness decreased and hebecame more unsteady.

On admission he was a pubertal boy with an unusual facial appearance and low hair line. Thetexture of his hair was not notably abnormal. He was very drowsy and at times unable to con-centrate in tests, and spoke with a cerebellar dysarthria. Apart from bilaterally impaired auditoryacuity, cranial nerve function was normal. The limbs were hypotonic and showed cerebellar ataxia.Power and sensation were normal. Tendon reflexes were depressed. Abdominal reflexes werepresent and plantar responses were flexor. His gait was very unsteady and he could barely walkwithout aid. Chest and abdomen were normal. His blood pressure was 100/60.

He hyperventilated intermittently. Within a few hours he sank into a comatose state.jDuringthe following day his level of consciousness fluctuated: at times he was unrousable, with abnormalepileptiform movements of his head and arms.

Initial investigation showed normal blood sugar, serum electrolytes, urea (25 mg./lOO ml.),cholesterol (220mg./100ml.) hsmoglobin and white cell count; negative blood Wassermannreaction; normal X-rays of chest and skull; and normal electrocardiogram. An electroencephalo-gram (fig. 2) was grossly abnormal, with generalized medium and high voltage slow wave activityand brief synchronous bursts of high voltage slow waves. Some of the changes were epileptic, andthe record suggested diffuse degenerative disease. Otological examination showed bilateralimpairment of caloric responses and moderate perceptive deafness.

FIG. 2.—Electroencephalograph recorded a day after admission whilstsemiconscious.

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ARGININOSUCCINIC ACIDURIA 415

On the third day he was given 120 mg. of phenobarbitone and 200 mg. of phenytoin over aperiod of ten hours, and his epileptiform movements disappeared as his coma deepened. Furtherinvestigation showed normal serum caeruloplasmin; calcium 9-3 mg./lOO ml. and phosphate51 mg./100ml.; normal plasma proteins and flocculation tests of liver function; bilirubin1 -5 mg./100 ml.; raised serum alkaline phosphatase (28 King Armstrong units/100 ml.) and raisedserum transaminases (SGOT 52 units; SGPT 98 units/ml.). The blood urea concentration wasnow 53 mg./lOO ml.

Because of respiratory difficulties, tracheostomy was performed on the following day. On themorning after operation he was found to be unresponsive to painful stimuli and to have bilateralsurgical emphysema. A chest X-ray showed bilateral pneumothorax. Before appropriate treatmentcould be begun his heart stopped, for an estimated ninety seconds, and was restarted with the helpof intravenous adrenaline and calcium gluconate. The pleura! cavities were drained of air bysuction. Spontaneous respiration was not established after his cardiac arrest, and he remained on arespirator, unresponsive to painful stimuli, with fixed dilated pupils and absent deep and super-ficial reflexes, until his death thirty hours later.

BIOCHEMICAL FINDINGSThe diagnosis was made by the detection of argininosuccinic acid in the urine and plasma.

Two-way chromatography of urinary aminoacids on thin places coated with MN-cellulose 300 GIn each of two solvent systems showed ninhydrin-positive spots corresponding to argininosuccinicacid and one of its anhydrides. Argininosuccinic acid, prepared from barium argininosuccinate(Sigma) by precipitation of the barium with potassium sulphate solution, was used as a referencecompound. In solution, anhydrides form spontaneously on standing, the rate of conversiondepending on pH and temperature (Ratner et al., 1953). The large amounts of argininosuccinicacid present in the urine of this patient made it possible to obtain satisfactory chromatogramsusing as little as one microlitre.

Plasma aminoacids were extracted by passing an 0-5 ml. sample of plasma through an Amberlite120 (H) column. The aminoacids were eluted with 5M ammonium hydroxide solution: the eluatewas taken to dryness and the residue redissolved in 0-1 ml. of distilled water. One-way chromato-graphy on MN-cellulose 300 G thin-layer plates, using butanol-acetic acid-water (4:1:1) as asolvent system and a 2 to 5 microlitre sample was used to demonstrate the presence of arginino-succinate in this plasma extract.

To confirm that argininosuccinic acid was present in the patient's urine its barium salt wasisolated by Westall's method (Levin et al., 1961). A pale yellow hygroscopic powder was obtained;a solution containing 20 mg./ml. was then prepared and barium was removed by the addition ofa drop of 0-6 N sulphuric acid and centrifugation. An aliquot of the supernatant Was at oncesubjected to two-way chromatography using butanol-acetic acid-water and phenol-ammonia asthe solvents. A 20 mg./ml. solution of barium argininosuccinate (Sigma) was used as standard, andwas treated in an identical way. Chromatograms from the two solutions each showed a singleninhydrin-positive spot with identical Rr values in the two solvents.

It was shown subsequently that barium argininosuccinate isolated from the urine could besatisfactorily substituted for the reference compound as the substrate in an assay system forargininosuccinase (L-argininosuccinate lyase, E.C.4.3.2.1.). This was considered to provide adequateconfirmation of the presence of argininosuccinate in the urine, and thus of the diagnosis.

Since no full twenty-four-hour urine collection was made, no direct determination of dailyargininosuccinate output was possible. However, the yield of barium argininosuccinate from arandom urine sample of known creatinine content suggested that the daily excretion of arginino-succinic acid was not less than 10 grammes.

The death of the patient made it possible to obtain a sample of liver adequate to study the fiveurea cycle enzymes (fig. 3). The activities of these enzymes were determined by the methods of

416 P. D. LEWIS AND A. L. MILLER

ENZYME ACTIVITY

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IUREA CYCLE ENZYMES

FIG. 3.—Liver urea cycle enzymes in argininosuccinic aciduria. The blocks indicate the activitiesof the different enzymes expressed as a percentage of the mean control value. The vertical barsindicate the ranges observed in five human control liver samples. A=carbamoyl phosphatesynthetase; B=ornithine transcarbamoylase; C=argininosuccinate synthetase; D=arginino-succinase; E=arginase.

(1) Ammonia+bicarbonate+2ATPinorganic phosphate.

(2) Carbamoyl phosphate+ornithine •

Simplified Urea Cycle

Carbamoyl phosphate Isynthetase

Ornithine

carbamoyl phosphate + 2ADP +

(3) Citrulline+aspartate+ATP-phosphate.

transcarbamoylase

Argininosuccinate

-»• citrulline+inorganic phosphate.

synthetaseargininosuccinate + AMP + inorganic pyro-

Argininosuccinase(4) Argininosuccinate >• arginine+fumarate.

Arginase(5) Arginine+H,0 »• urea+ornithine.

Brown and Cohen (1959) as modified by McLean and Gurney (1963). The studies showed a markeddecrease in argininosuccinase, approximately 3 per cent of normal activity being present (Millerand McLean, 1967). The activities of other enzymes of the cycle were normal by comparison withother post-mortem liver samples.

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ARGIN1NOSUCCINIC ACIDURIA 417

The urine of the patient's parents and their surviving siblings was examined by one-waychromatography. No argininosuccinic acid was detected.

MORBID ANATOMICAL FINDINGSNecropsy was carried out twenty hours after death.The brain was swollen, with flattening of gyri and narrowing of sulci, but was otherwise nonnal

externally. Coronal slicing after fixation showed no macroscopic abnormality, the cerebral cortexbeing of nonnal thickness and colour. Brain-stem, cerebellum and cervical spinal cord appearednormal macroscopicalry.

The liver was enlarged (2,505 grammes) with a smooth external surface and a pale yellowish cutsurface showing a normal lobular pattern.

The lungs were normally inflated, bronchi being patent. The lower lobes were congested. Surgicalemphysema was present in the mediastinum.

The pituitary, thyroid, adrenals, heart, kidneys, spleen, pancreas, stomach and intestinesappeared normal.

Microscopy.—Paraffin and celloidin sections of the brain and spinal cord were stained withhaematoxylin and eosin, Luxol fast blue, cresyl violet and Holzer's method for glial fibres. Frozensections of cerebrum were stained with Sudan HI.

In the cerebral cortex neurones showed minor anoxic damage (ischaemic nerve cell change ofSpielmeyer) and post-mortem change, but their number was normal. Interspersed with normalastrocytes, especially in the deeper part of the cortex, were scattered large glial nuclei with clearmembranes, watery nuclear sap and scanty chromatin, and sometimes a prominent nucleolus,resembling Alzheimer type II cells (fig. 4). These were seen in all cortical regions. The number ofsubconical glial nuclei was increased, but there was no increase of glial fibre density. No corticalnecrosis was seen. Oligodendrocytes appeared normal in the subcortical white matter, and themyelin of the cerebral hemispheres was normal. No microglial proliferation was observed.

FIG. 4.—Abnormal astrocytic nucleus (Alzheimer type IT) in cerebral cortex.H.E. X1280.

418 P. D. LEWIS AND A. L. MILLER

The thalamus was of normal size but grossly depleted of neurons. In some areas only a fewghost cells and very damaged neurons remained. The neuronal loss was most severe anteriorly andaffected particular nuclear groups. Except for anterior dorsalis (in the nomenclature of Dekaban,1953) all anterior nuclei were severely damaged, as* was the smaller-celled part of medialis dorsalisand the entire ventral group save the posterior part of ventralis lateralis. The centrum medianumwas almost devoid of neurons. In contrast the mid-line nuclei were spared, showing strikinglywell-preserved neurons (fig. 5). Some pallor of thalamic myelin was noted.

FIG. 5.—Thalamus. Haematoxylin and eosin. x320. A, Parafascicular nucleus, B, Centrummedianum, showing loss of neurons.

There was neuronal loss in pulvinar, geniculate bodies, red nucleus and corpus luysii. The hypo-thalamus, caudate, putamen and pallidum presented only minor anoxic change, with a normalnumber of neurons. Even in the most devastated areas there was no gliosis. Alzheimer type IIcells were seen in all these parts, but were no more numerous in the thalamus than elsewhere. Noastrocytic nuclei corresponding to Alzheimer type I cells were found. Neuronal damage was seenin substantia nigra, in which region pigment-containing macrophages were present.

Sections of mid-brain and pons showed no other significant abnormality. Neurons in the cere-bellum showed minor anoxic and post-mortem change, but were normal in number. Abnormalastrocytic nuclei were present in the dentate nucleus.

ARGININOSUCCINIC ACIDURIA 419

Sections of the medulla through the caudal third of the olive showed normal vestibular andcochlear nuclei. Fibre tracts in the brain-stem were normal. The anterior horns in the uppercervical cord contained normal neurons interspersed with occasional abnormal astrocytes.

The liver architecture was normal, with increased fat deposition. Kidneys showed post-mortemchange. Adrenals, pancreas and spleen were normal.

DISCUSSION

The clinical features of argininosuccinic aciduria are shown typically by the presentcase. Nearly all reports describe severely retarded children whose symptoms haveappeared early in life. Most have suffered from epilepsy, generally with electro-encephalographic abnormality, and a proportion have been ataxic: as in our patient,ataxia has been noted to be intermittent. As occurred in the present case, an episodeof short-lived coma associated with a very dysrhythmic EEG has been described(Moser et ah, 1967). This episode was accompanied by hyperammonasmia, theimplications of which will be discussed. Some reported cases have had abnormal,friable hair. Clinical hepatomegaly has been noted in several instances. In a numberof patients liver function tests—notably alkaline phosphatase and SGOT—havebeen abnormal.

The diagnosis has been made by the detection of argininosuccinic acid in the urineand in some cases in blood and cerebrospinal fluid. Autosomal recessive inheritanceis likely: examination of the red blood cells for reduced argininosuccinase activity,as suggested by Tomlinson and Westall (1964), might be a method of detectingheterozygotes.

From the biochemical viewpoint, argininosuccinic aciduria presents two interest-ing problems. The first is that in all reported cases the blood urea and the rate of ureaproduction have been normal, despite a proven defect in the Krebs-Henseleit ureacycle. This has led to the supposition that alternative pathways exist for the synthesisof urea (Levin et ah, 1961), and the theory that hepatic argininosuccinase activitymight be normal in this condition, defective activity of isoenzymes in other tissuesbeing responsible for the accumulation of argininosuccinic acid (Allan et ah, 1958).Our finding of a marked reduction in the hepatic argininosuccinase activity in apatient who died of argininosuccinic aciduria excludes the latter possibility, and alsomakes it unnecessary to postulate the existence of alternative pathways of ureasynthesis since the observed residual enzyme activity was capable of supporting thesynthesis of a calculated maximum of 0-5 mole of urea per day. Moreover, Craneet ah (1969) have shown normal urea production from labelled ammonia in thiscondition.

The second problem is the cause of the clinical manifestations of this metabolicdisorder. One obvious possibility is that argininosuccinate, normally a transientintermediary compound, is directly toxic to certain cells or metabolic processes whenit accumulates. Direct evidence for any such toxic action is lacking, however. Alterna-tively, the clinical effects might be due to chronic arginine deficiency, since it may bethat endogenous arginine production by the cleavage of argininosuccinate is a

4 2 0 P. D. LEWIS AND A. L. MILLER

major function of the urea cycle, in some tissues at least. Certainly endogenousarginine synthesis can be shown experimentally to have a physiological role (Schimke,1962; McFarlane, 1963). Another possibility is that argininosuccinate may beincorporated into physiologically important proteins in place of arginine, withdisturbance of function. Shelley and Rawnsley (1965) reported the presence ofargininosuccinate in the hair of a patient—a condition which they called "amino-genic alopecia"—and suggested that this was due to the substitution of arginino-succinate for arginine in keratin. We have examined hair hydrolysates from apatient with argininosuccinic aciduria and, in contrast, could detect no arginino-succinate.

Probably the most likely explanation of the symptoms and signs in this condition ishyperammonaemia, which has been demonstrated by Moser et al. (1967) as notedearlier. The neurotoxicity of high concentration of blood ammonia has been sus-pected for some years (Bessman and Bessman, 1955), and in argininosuccinicaciduria—as distinct from liver cell failure—the problem of non-detoxication ofother substances does not arise. Ammonia might well damage other tissues, includingthe liver.

The most important neuropathological feature of the present case is the findingof a number of atypical astrocytic nuclei, similar in appearance to cells first describedin a case of Wilson's disease by von Hosslin and Alzheimer in 1912, subsequentlynamed Alzheimer type LT cells, and now regarded as a consistent finding in the brainsof patients with severe chronic liver disease (Victor et al., 1965). Their distribution incases of hepatic coma parallels that seen in our patient, though other abnormalitiesdescribed in chronic hepatocerebral degeneration by Victor et al.—notably corticallaminar necrosis and polymicrocavitation—are absent in the present case.

Although the exact relationship of hepatic encephalopathy to hyperammonaemiahas for long been uncertain, it has recently been firmly established that the cardinalneuropathological change in this liver disorder—the appearance of Alzheimer type IIcells—is dependent on an increased amount of blood ammonia. Cavanagh andKyu (1970) have shown the correlation of abnormal astrocyte number withthe plasma ammonium-nitrogen level in rats a month and more after porto-cavalanastomosis.

Furthermore, in a child who died of hyperammonffimia due to ornithine trans-carbamoylase deficiency (Russell et al., 1962), the cardinal pathological feature isthe presence of large numbers of Alzheimer type II cells (Bruton, Corsellis andRussell, 1970). Presumably their appearance is a direct consequence of ammoniatoxicity. The comparatively small number of these cells in our case points toa relatively low level of blood ammonia, and in fact the levels in the patientsstudied by Moser et al. (1967) were only half or less of those in Cavanagh and Kyu'sexperimental animals.

Apart from abnormal astrocytes, a striking neuropathological finding was grossneuronal loss in the thalamus. The pattern of nuclear damage seemed random, though

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ARGININOSUCCIN1C ACIDURIA 421

it is apparent that the bulk of the "neothalmus" was ravaged while the primitive,mid-line nuclei survived virtually intact. This loss of neurons, in the presence of anormal-sized thalamus and the absence of gliosis, suggests the possibility thatterminal anoxic damage and post-mortem autolysis might be responsible, for if thenuclei were depleted from an early age the thalamus would be small, while if theneuronal loss were chronic some gliosis might be expected. However, it may berelevant that in hepatic coma there is a lack of fibrous glial reaction (Mossakowski,1966). The colchicine-like inhibitory action of ammonia on mitosis (Cavanagh andKyu, 1969) might also be implicated here, for astrocytic proliferation in response toneuronal damage would be prevented: however, the absence of Alzheimer's type Icells, as demonstrated by Cavanagh and Kyu, might be held as a point against thisargument.

In our retarded patient, the possibility that this localized loss of neurons was notan artefact is suggested by the observation that dementia, sometimes of severe degree,may result from circumscribed thalamic lesions (Smyth and Stern, 1938), probably onaccount of the exclusive projection of the dorsomedial nucleus to the frontal cortex.With the aim of making a retrospective diagnosis of argininosuccinic aciduria, somepublished case reports of chronic "thalamic dementia" and thalamic damageassociated with long-standing epilepsy (Grtinthal, 1942; Scholz, 1951) wereexamined. None which corresponded clinically or pathologically to the presentcase could be found.

At the present time, no firm conclusions can be drawn about the thalamic lesionsin this case. The one other autopsied case (Baumgartner et al., 1968), who died afterfour days of convulsions and coma, showed a very different picture. The brain wasoedematous. The cerebral cortex was depleted of neurons and showed mild glialproliferation, spongy rarefaction of cerebral white matter, and microcysts in thecortex and central grey matter. Some of these changes are found in infants sufferingfrom other primary aminoacidurias, notably phenylketonuria and maple syrupurine disease (Crome and Stern, 1967).

SUMMARY

A mentally retarded 16-year-old boy presented with episodes of somnolenceassociated with ataxia and epilepsy. His urine contained large amounts of arginino-succinic acid, and post-mortem study of liver enzymes showed marked deficiency ofargininosuccinate lyase.

Abnormal astrocytes resembling Alzheimer type II cells were found in the brain,and there was gross loss of neurons from the thalamus. The possible interrelation ofhyperammonffimia, clinical features and pathological changes is discussed.

The authors arc indebted to Professors J. B. Cavanagh and H. Urich and to Dr. M. Kremer,under whose care this case was admitted.

422 P. D. LEWIS AND A. L. MILLER

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(Received 22 October 1969)

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