Deselop. Med. Child Neurol. 1963, 5, 586592

Abnormal Amino-Acid Excretion in Cerebral Disease

Neil Gordon Vera K . Wilson

AT this hospital, during the routine investigation of urinary amino-acids by paper chromatography, one of us (V.K.W.) found an unknown ninhydrin reacting substance in the urine of a patient suffering from convulsions, mental retardation and ataxia. The same substance was also found to be excreted by a sibling. A report of the cases was published by Allen et al. (1958). Later this unknown substance was identified by Westall (1958) as arginino- succinic acid and the name ‘arginino- succinic-aciduria’ given to the syndrome.

It was then decided to investigate the amino-acid urine excretion of as many children as possible with mental deficiency, epilepsy and other evidence of cerebral disease. Although no other case of argininosuccinic aciduria has been dis- covered, it was soon apparent that most of these children had an abnormal amino- acid pattern, chiefly related to the amino- acids containing sulphur.

Materials and Methods Two-dimensional chromatography was

used for all abnormal cases, using the method of Dent (1948). The volume of urine applied contained 250 pg. of total nitrogen for large squares, 200 pg. for medium squares and 150 pg. for smaller

squares. The squares were run in phenol as the first solvent and then in 80 per cent aqueous tetrahydro-furfurol alcohol as the second solvent (Bentley and Whitehead 1950). Every specimen was analysed, oxidised to identify cystine and methionine, and unoxidised. Both squares were sprayed with 0.1 per cent ninhydrin in n-butanol and the depth of colour obtained was measured on an arbitrary scale of 10 units, as suggested by Dent (1947).

In cerebrospinal fluid analyses, the fluid was deproteinised by ultrafiltration and electrolytically desalted ; 625 PI. was then taken for the large squares and 500 p1.

for the medium squares. Again two squares were run in each case, oxidised and unoxidised.

Findings in Normal Children A normal urine amino-acid chromato-

gram from a child over three weeks old has shown, using our method, the following amino-acids : serine, glycine, alanine and glutamine. The dominant amino-acid in the urine is always glycine, usually of strength 6 to 7 in our arbitrary units. Occasionally a trace of histidine and p-aminoisobutyric acid is to be found. The latter is known to be excreted by about 5 per cent of normal subjects.

_- - ~ _ _ ~ _ _ _ _ Royal Manchester Children’s Hospital, Pendlebury, Lancashire.

586

NEIL GORDON VERA K . WILSON

Using the volume of urine corresponding to 250 pg. of total nitrogen, we have not found any taurine in the urine of normal children. The one-way chromatogram in phenol was judged to be abnormal when any bands were present corresponding to amino-acids other than the normal ones; and when the depth of colour of normal amino-acid bands indicated that the quantity present was definitely in excess.

When no such changes were found only one-way chromatograms were done, in most cases on urine containing 250 pg. of total nitrogen.

Results in Cerebral Disease The patterns obtained in cases of cerebral

disease could be classified into the follow- ing groups:

1. Cystine only in excess. 2. Cystine and glutamic acid in excess. 3. Glutamic acid in excess. 4. Cystine and taurine in excess. 5. Cystine, taurine and glutamic acid in

excess.

The results are shown in Table I. Of the 219 urines examined, 45 were normal, 69 showed cystine only in excess, 33 showed cystine and glutamic acid in excess, 11 showed glutamic acid in excess, 46 showed cystine and taurine in excess and 15 showed cystine, taurine and glutamic acid in excess. The results are given as per- centages in Table 11. Although no control group was included in the study such findings were rare when the examination was done for other diseases.

Serial Examination of Urinary Amino-Acid Chromatograms

The urines of 24 abnormal cases were investigated more than once, 19 being done twice, 4 three times and 1 on four occasions. These patients’ urines showed an almost identical pattern on each occasion, suggesting that the abnormalities

were not merely transient but represented a persistent disorder.

The Presence of Glutamic Acid The finding of glutamic acid in a

chromatogram always leads to controversy since it may have arisen as a decomposition product. However, specimens were always collected in the early morning and imme- diately placed in a refrigerator, using a crystal of thymol as a preservative, and the analyses were usually done on the same day, so it is unlikely that the com- pound arose from decomposition.

Cerebrospinal-Fluid Analyses The results of the cerebrospinal fluid

analyses are reported in Table 111. A specimen of urine from the patient was collected if possible on the same day as the lumbar puncture was done or very soon afterwards. Although large amounts of cystine were found in some of the urine specimens no cystine was found in the same patient’s cerebrospinal fluid. There seemed to be little correlation between the chromatogram pattern in the urine and in the cerebrospinal fluid. None of the cerebrospinal fluids analysed contained p-aminoisobutyric acid, cystine, histidine, taurine, phenylalanine or y-aminobutyric acid. Alanine, glycine, glutamine, leucine, serine, threonine and valine were present in over 80 per cent of the cases. Aspartic acid, glutamic acid, lysine, tyrosine and arginine were less frequently found. As in the study of Walker et a/. (1955), no diagnostic pattern of abnormal amino- acids in the cerebrospinal fluid has been demonstrated. These authors consistently found y-aminobutyric acid in the cerebro- spinal fluid, but our results agree with those of Logothetis (1955), who did not confirm this finding.

The Effect of Treatment on Amino-aciduria Among the patients with these various

587

DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1963, 5

Cystine and

glutamic acid in excess

TABLE I Chromatogram Patterns Found among the 219 Urines Examined

(The number expresses the value in arbitrary units of the amino-acid and the numbers in brackets represent the range of values obtained.)

Glutamic acid in excess

I I Cystine and

taurine in excess

Cystine, taurine

and glutamic acid in excess

Normal Amino-acid Cystine in excess

5 (2 - 9)

-

Alanine .. .. .. 5 I (3 5_ 7) I (2 - 8) 4 5 5

(2 - 5)

0.6 (0 - 4)

(3 - 7) I (2 - 6)

-

Glutamine * . . . .

Histidine . . . . . .

Aspartic acid . . . . 1 - l -

4 - I (0 - 8)

0.4 2 (0 - 3) I (0 - 6)

I -

Valine . . . . . .

No. of cases . . . . TOTAL .. .. ..

/ (:!6) 1 (0 3 8) PAminoisobutyric acid . .

0.6 (0 - 6) -

45 69

3 - 1 (0 f! 5) 1 (0 4_ 9) ! (0 - 8)

0.6 (2 - 8)

33

Cystine . . . . . . 5 (as cysteic acid) I - l ( 3 - 8 )

0.8 0.5 - (0 - 7) (0 - 6)

1 1 46 15

5 0 .3 5 (2 - 7) 1 (0 - 3) I (3 f 8) I (3 - 7)

Glycine . . . . . . 7 ! (5 8) I (5 - 8) 7 7

(5-10) I (4 6 7) I (6 8) I (3 - 9)

Glutamic acid .. . . I - i - 5 5 (2 - 8) i (2 1. 8) 1 - (3 - 8)

1 2 3 (0 - 6) I (0 1 3 ) (0 - 6) ~ (0 - 5 )

0.4 (0 - 6)

(0 - 4)

-

0.6 0.5 (0 - 5)

Lysine . . . . 0.2

Leucine .. .. (0 - 4)

-

Methyl histidine . . . . ! - I - 4 4

(0 - 6) I (0 3 6) i (0 I 7) I (0 - 8)

Taurine .. .. .. I - l - ~ ~~

Threonine . . . . . . 1 I - l ( 0 - 6 )

Tyrosine . . . . - j - I -

1 219

* One-way chromatograms were done for most of the normal urines, so the glutamine spot would overlap the alanine.

588

NEIL GORDON VERA K. WILSON

Average arbitrary Amino- Acid measurement of depth

of colour when present

TABLE I1 Relative Frequency of Chromatogram Patterns in the 219 Specimens of

Urine Examined

Percentage frequency of appearance

Chromatogram pattern I per cent

I Leucine :: I: 1 4 Serine . . 6

~~

Normal . . . . . . . . . . . . Cystine in excess . . . . . . . . . . Cystine and glutamic acid in excess Glutamic acid in excess . . . . . . . . Cystine and taurine in excess Cystine, taurine and glutamic acid in excess . .

. . . . . . . . . .

100

88

20.6 31.5 15.1 5.0

21.0 6.8

Tyrosine . . . . 1 2

Valine . . . . . . 4

TABLE 111

of Cerebrospinal Fluid Examined Amino-acid Chromatogram Patterns found in the 17 Specimens

12

100

r Alanine . . :: 1 5 100

Aspartic Acid 4

I Glycine . . . . 1 5 I 100

. . . . 12 I Lysine.. 1 2 I

. . . . 82 I Threonine 1 5 I

1 Arginine . . . . 1 5 I 12

types of abnormal amino-aciduria, about barbiturates. From these figures there a third were known to be on treatment at seemed to be no relationship between any the time the specimen of urine was of the abnormalities found in the urine examined, except in the group excreting and the fact that the patient might or an excess of cystine and taurine, where might not be taking drugs of this kind. the proportion was nearer 50 per cent. With few exceptions this treatment was Amino-aciduria in Patients with Seizures being given to control epileptic seizures and Mental Retardation and consisted of phenobarbitone and Table IV showed that the different occasionally primidone or phenytoin chromatogram patterns occur in a roughly sodium. Only about a quarter of the similar proportion of patients suffering patients with normal urinary chromato- from epilepsy and from mental retarda- grams for amino-acids were on treatment, tion. There is nothing to suggest that the but again in most cases this consisted of presence or absence of convulsions has

589

DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1963, 5

Number of Chromatogram Findings patients in

each group

TABLE IV

Incidence of Epilepsy and Mental Retardation among 219 Patients with Various Abnormalities of the Urinary Chromatograms for Amino-Acids

Number with

epilepsy

Number with mental retardation

21 ~~

Normal . . . . . . I 45 I 16

Number with mental

retardation an& epilepsy

4

Cystine in excess . . . . 1 69 1 15

Cystine and taurine in excess

Cystine and glutamic acid in excess . . . . . .

46 14

33 10

14

14

any direct relationship to these findings, and so far there has been no correlation between the chromatogram results and the recorded abnormalities on examination of the mentally retarded patients. It is therefore impossible to draw any definite conclusions on the nature of the abnor- malities found in these patients’ urines, except to suggest that the disorder of metabolism may be of a non-specific type.

6

3

Discussion Harris (1949) rightly emphasised that

the presence of an amino-aciduria does not necessarily imply a primary defect of amino-acid metabolism. The serum amino- acid levels were not estimated in our study, but Choremis, Kyriakides and Karpouzas (1959) found normal levels in epileptics showing a similar amino-aciduria, which does not suggest that this is an ‘overflow’ amino-aciduria such as is found in phenylketonuria. The possibility that the findings are due to a ‘renal amino-aciduria’ has to be considered, although it would seem unlikely that in such a diverse group of patients a defect of renal tubular re- absorption could be the entire explanation of the excess of amino-acids in the urine.

Cystine, taurine and

Glutamic acid in excess

glutamic acid in excess . . . .

15 4

1 1 4

27 1 10

3

2

2

2

Some amino-acids may not be adequately reabsorbed in the presence of normal renal function because they do not usually occur in appreciable amounts in the blood. This has been referred to as a ‘no-threshold’ amino-aciduria (Allan et al. 1958). There is evidence that in certain metabolic cerebral disorders, particularly those re- sulting from pyridoxine deficiency, amino- acids are in fact excreted in abnormal amounts. Pyridoxal-5-phosphate is the co-enzyme for a number of enzymes concerned with the metabolism of sulphur- containing amino-acids, and Hope (1958) has studied the effects of pyridoxine deficiency in two of these, taurine and cystathionine. Blaschko et al. (1953) reported that taurine disappeared from the urine of pyridoxine-deficient rats, but later studies showed that in fact it con- tinued to be excreted and that its source was most probably nervous tissue. Cysta- thionine also appeared in the urine of these rats, and experiments strongly suggested that the major part of this substance was produced by the brain as well (Hope 1958).

Bickell (1 952) examined the urinary chromatograms for amino-acids from 200

590

NEIL GORDON VERA K. WILSON

normal children and found that 4 to 6 amino-acids were usually present, most commonly glycine, alanine and glutamic acid, and more rarely glutamine, histidine, cystine and taurine. His analyses were based on 500 pg. of total nitrogen excretion in the urine, and only small amounts of the rarer amino-acids, such as taurine, were found. This is perhaps why taurine was never found to be a normal excretion product in children during the present study, which used 250 pg. of total nitrogen excretion, although it was often present in excess in the abnormal amino- acid chromatograms. Cystathionine has not so far been found in the urines of any of the patients under review. However, Harris (1949) found it in the urine of an elderly imbecile and demonstrated that its excretion was markedly increased by giving the patient methionine. There is in fact no indication of the metabolic abnormalities that may underly our find- ings, but their high incidence in children with cerebral disease suggests that they may reflect a disorder in the synthesis of amino-acids by the brain cells. It is hoped that further studies on tryptophan deriva- tives may throw more light on this problem.

Choremis, Kyriakides and Karpouzas (1959) found increased levels of amino- acids in the urine, with normal levels in the serum, in 13 out of 16 patients during convulsions, and the abnormali t ies appeared to be related to the severity of the seizures. These results are also not strictly comparable to the ones recorded in this paper, the analyses being based on 500 rather than 250 pg. of the total nitrogen urine excretion, although the pattern of cystine, taurine and glutamic acid excretion was similar in the two groups. As has been mentioned, there did not appear to be any definite relationship between our findings and the presence or absence of epileptic seizures, and in a

C 59 1

more recent study by Choremis, Kyriakides and Basti-Maounis (1961) no correlation was demonstrated between an abnormal amino-aciduria and the incidence of ‘infantile spasms’. These authors found an increased amino-aciduria in 9 out of 11 mentally retarded children suffering from such ‘spasms’. The amino-acids most commonly found in excess were alanine, cystine, glycine, glutamic acid, histidine, leucine and threonine. The abnormal pattern remained when the patients were free of attacks, and although steroids led to clinical and electroencephalographic improvement, there was no immediate effect on the chromatogram. Von Gelderen and Dooren (1963) measured the free alpha-amino-nitrogen excretion in 13 1 mentally retarded children and found that the incidence of abnormal amino-acidurias was 27 per cent among the group of familial severe oligophrenias without con- genital malformations. Their findings were not related to the anticonvulsant drugs which the children were taking.

This can be regarded only as a pre- liminary report published in the hope that it will interest other workers in this field. The intention is not to postulate any definite theory to account for the presence of certain amino-acids in these children’s urine but only to suggest that our results warrant further study. If pyridoxal-5- phosphate can act as a co-enzyme for a number of enzymes concerned with the metabolism of the sulphur-containing amino-acids, and if its deficiency can change the amounts of these substances in various tissues, it seems possible that a common cause might also underly some of these findings.

Acknowledgements: We are grateful to the con- sultants at the Royal Manchester Children’s Hospital for referring patients for these investiga- tions. We are much indebted to the organisers of the Manchester University Rag Fund for a research grant which enabled us to employ a technician. We are also grateful to Dr. Komrower for his advice on the presentation of this work.

DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY. 1963, 5

SUMMARY AND CONCLUSIONS An abnormal amino-aciduria has been found in 174 out of 219 children with evidence

of cerebral disease. No arginino-succinic acid was excreted in any case. In most patients it was the sulphur-containing amino-acids that were in excess. No correlation could be found between the amino-aciduria and the patient’s clinical condition. Treatment seemed to have no effect on the amino-aciduria. Similar patterns were obtained when the urine from individual patients was analysed on a number of different occasions. The cerebro- spinal fluid amino-acids showed no specific pattern.

RESUME et CONCLUSIONS ExcrPtion d’amino-acide anormal au cours de maladie cPrPbrale

Une amino-acidurie anormale a CtC dCcelte chez 174 enfants parmi 219 prtsentant des signes de maladie c6rCbrale. L‘acide arginine-succinique n’Ctait excrete en aucun cas. Les amino-acides en ex&s consistaient chez la majorite des patients en amino-acides contenant du soufre. I1 n’y avait aucune correlation entre l’amino-acidurie et l’Ctat du malade. Le traitement etait sans effet sur l’amino-acidurie. Les analyses de l’urine du mCme patient faites B differentes occasions ont donne les mCmes rksultats. Le liquide ctphalo-rachidien n’a pas present6 de formule spkcifique.

ZUSAMMENFASSUNG Abnormale Aminosauren-Ausscheidungen bei Zerebrallahmung

Bei 174 von einer Gesamtzahl von 219 Kindern mit erwiesener Zerebrallahmung fand man Aminosauren im Harn. Bei keinem der Patienten war eine Ausscheidung von Succinoarginisaure erweisbar. Das tibermass der Aminosauren war bei den meisten Patienten in der Form von Aminosauren enthaltendem Schwefel. Es gelang nicht eine Korrelation zwischen dem klinischen Zustand und den Aminosauren im Harn festzustellen.

Ahnliche Schablonen fand man, wenn man den Harn einzelner Patienten zu verschiedenen Zeitpunkten analysierte. Die Aminosauren im Liquor folgten keiner bestimmten Schablone.

Allan J. D., Cusworth, D. C., Dent, C. E., Wilson, V. K. (1958) ‘A disease, probably hereditary, charac- terised by severe mental deficiency and a constant gross abnormality of amino-acid metabolism.’ Lancet, i, 182.

Bentley, H. R., Whitehead, J. K. (1950) ‘Water-miscible solvents in the separation of amino-acids by paper-chromatography.’ Biochem. J., 46, 341.

Bickel, H. (1952) Amino-aciduria in Childhood. Univ. of Birmingham: Ph.D. Thesis. Blaschko, H., Datta, S. P., Harris, H. (1953) ‘Pyridoxin deficiency in the rat. Liver L-cysteic acid decarb-

oxylase activity and urinary amino-acids.’ Brit. J. Nutr., 7 , 364. Choremis, C., Kyriakides, V., Basti-Maounis, V. (1961) ‘144: Amino-acidurie in infantile spasms with

hypsarhythmia.’ 7th Int. Congr. Neurol. Excerpta Medica Int. Congr. Series, No. 38, 144. - - Karpouzas, J. (1959) ‘Amino-aciduria in epilepsy.’ J. Pediat., 55, 593. Dent, C. E. (1947) ‘The amino-aciduria in Fanconi syndrome; a study making extensive use of techniques

based on paper partition chromatography.’ Biochem. J., 41, 240. - (1948) ‘A study of the behaviour of some sixty amino-acid and other ninhydrin-reacting substances

on phenol-“collidine” filter-paper chromatograms, with notes as to the occurrence of some of them in biological fluids.’ Ibid, 43, 169.

Harris, H. (1959) Human Biochemical Genetics. London: Cambridge University Press. Hope, D. B. (1958) ‘Studies of taurine and cystathionine in brain.’ 4th Int. Congr. Biochemistry, I.U.B.

Symposium Series, ed. 0. Hoffman-Ostenhof, vol. XIII, COIL 63. Logothetis, M. D. (1955) ‘A study of free amino-acids in human cerebrospinal fluid.’ Neurology, 5, 767. Von Gelderen, H. H., Dooren, L. J. (1963) ‘Studies in oligophrenia. 11. Amino-aciduria in mentally

deficient children.’ Acta paediat. (Uppsala), 52, 41. Walker, B. S., Telles, N. C., Pastore, E. J. (1955) ‘Amino-acids of the,cerebrospinal fluid; normal paper

chromatographic pattern and its duplication in multiple sclerosis. Arch. Neurol. Psychiar. (Chic.),

REFERENCES

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form of mental deficiency.’ In Biochemistry of Lipids, Proc. 5th Int. Conf. on Biomechanical Problems of Lipids held at 4th Int. Congr. of Biochemistry, Vienna, ed. G. Popjak.

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