Archives of Disease in Childhood, 1986, 61, 130-137

Neurological aspects of biopterin metabolismI SMITH, R J LEEMING, N P C CAVANAGH, AND K HYLAND

Hospital for Sick Children and Institute of Child Health, London, and The General Hospital, Birmingham

SUMMARY Plasma total biopterin concentration was measured by bioassay in 59 infants withhyperphenylalaninaemia and in 50 children with developmental regression and or movementdisorder with normal plasma phenylalanine concentrations. In infants with raised phenylalanineconcentrations plasma biopterin concentrations were significantly raised in proportion to thephenylalanine values. Five patients had plasma biopterin concentrations at the extremes of therange, and of these two had defective biopterin metabolism. One with low plasma biopterinconcentration apparently had a partial defect of biopterin synthesis but died before investigationswere complete. One with high plasma biopterin concentration, even when phenylalanineconcentrations had fallen to the normal range, had dihydropteridine reductase deficiency. In thispatient concentrations of homovanillic acid and 5-hydroxyindolacetic acid in the cerebrospinalfluid (CSF) were severely reduced.

In children without hyperphenylalaninaemia plasma biopterin concentrations were normal.Twenty two patients were subjected to lumbar puncture, of whom six with developmentalregression without movement disorder had normal CSF biopterin concentrations, and 11 withmovement disorder other than torsion dystonia had significantly lower CSF biopterinconcentrations. Five patients with torsion dystonia had normal biopterin concentrations.

Tetrahydrobiopterin is the essential cofactor forthree hydroxylation reactions, the conversion ofphenylalanine to tyrosine, tyrosine to L-dopa, andtryptophan to 5-hydroxytryptophan. ' These last tworeactions are the rate limiting steps of catecholamineand serotonin synthesis. Deficiency of tetrahydro-biopterin, due either to defective biopterin synthesisfrom guanosine triphosphate or dihydropteridinereductase deficiency (Fig. 1), is the cause of hyper-phenylalaninaemia in 1-3% of infants detected onroutine neonatal screening.25 Children with de-ficiency of tetrahydrobiopterin may develop pro-gressive neurological disease, in which movementdisorder is a prominent feature and which isunresponsive to a low phenylalanine diet and due inlarge part to deficiency of neurotransmitter amines.We wished to assess whether measurement ofplasma total biopterin concentrations would help indistinguishing patients with hyperphenylalani-naemia due to defective biopterin metabolism fromthose with phenylalanine hydroxylase deficiency.Symptomless patients with normal plasma phenyl-

alanine concentrations, yet defective biopterinsynthesis, have been described,6 and others may

GTP

B, BH2 j

Ne 2P3 --- NeH2

Tyr 4-- q-BH2 NADPH 2

PH DHPR 3 2

Phe BH4 NADP 3

L..1

3

Fig. 1 Synthesis and recycling of tetrahydrobiopterin(BH4).GTP=guanosine triphosphate. NeH2P3=dihydroneopterin triphosphate,NeH2=dihydroneopterin, 'X' =tetrahydroketopterin, SeH2=sepiaterin, q-BH2=quinonoid-dihydrobiopterin, BH2=7, 8 dihydrobiopterin, B=biopterin,* = active in Crithidia fasciculata bioassay. Phe= phenylalanine, Tyr= tyrosine.Enzymes: PH=phenylalanine hydroxylase, DHPR=dihydropteridine reduc-tase, I=GTP cyclohydrolase, 2=tetrahydroketopterin synthesising enzyme,3=sepiapterin reductase.

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have symptoms, and/or phenylalanine accumula-tion, only when exposed to an excessive phenyl-alanine intake.7 It is not known how many childrenwith neurological disease may be suffering fromoccult disorders of biopterin metabolism, and partof the present study was to assess the prevalence ofsuch disorders among children with severe neurolo-gical disease, especially that accompanied by move-ment disorder. Adults with movement disorder mayhave reduced 'hydroxylase cofactor activity' incerebrospinal fluid (CSF),8 9 and there are reportsof impaired biopterin synthesis in the brains ofpatients with senile dementia. '1

Patients

Infants with phenylketonuria. Plasma was obtainedfrom 53 consecutive infants detected on routineneonatal screening before introduction of the lowphenylalanine diet. Seventeen were retested after afew weeks on treatment. Plasma was also obtainedfrom six infants who had already started the dietwhen first seen. Five infants underwent furtherinvestigation (see below), including lumbar punc-ture in one patient, because initial biopterin concen-trations were 'high' or 'low' (2SDs) compared withthe rest of the group.

Patients with neurological disorder. These were 50children aged 1 month to 16 years undergoingneurological investigation because of developmentalregression or extrapyramidal disturbance. Theywere investigated with the permission of theirparents and with the approval of the hospital ethicalcommittee. All had plasma phenylalanine concen-trations below 200 imol/l (3.3 mg/100 ml), and thepathogenesis of their illness remained obscure de-spite extensive investigation. Five patients hadtorsion dystonia, four had dominantly inherited tics,19 had developmental regression, accompanied byepilepsy in nine, and in a further 22 patientssevere extrapyramidal disturbance (choreoathetosis,tremor, dystonic posturing) was a prominent featureof their illness, accompanied by mental retardationin 13 and epilepsy in eight. Plasma was also obtainedfrom the four affected parents with dominantlyinherited tics.

Methods

Measurement of total biopterin activity. Heparinisedblood 1-2 ml was obtained by venepuncture, andplasma was separated by centrifugation. Between0-5 and 1 ml of CSF was obtained by lumbarpuncture using a standard technique. Specimenswere frozen as soon as possible and stored at -20(C.

Neurological aspects of biopterin metabolism 131

For analysis, samples were thawed, diluted with 0-2molar, pH 5-0 phosphate buffer 1:20, autoclaved,and assayed as described previously using Crithidiafasciculata.6 1 The growth response was readagainst a pooled plasma blank using standards oftetrahydrobiopterin. In two patients with inbornerrors of biopterin metabolism the crithidia resultswere confirmed by urine pterin analysis'2 (DrA Niederwieser, Zurich; Professor J A Blair,Birmingham) using high performance liquid chroma-tography. Urine contains roughly 1000 times theconcentrations of total biopterin compared withthose in plasma."'

Oral load of tetrahydrobiopterin. The protocol usedhere was that of Niederwieser et al.'3 The test iscarried out when plasma phenylalanine concentra-tions are raised. After an overnight fast 7-5 mg/kg oftetrahydrobiopterin combined with vitamin C (sup-plied by B Schircks, Zurich) was given on a spoon.Plasma phenylalanine and tyrosine concentrationswere measured at baseline and at one, two, four,six, eight, and 24 hours, and urine, or plasma,biopterin concentrations were measured to showthat the tetrahydrobiopterin had been absorbed.

Oral load of L-phenylalanine. After an overnight fast100 mg/kg of L-phenylalanine was given dissolved infruit juice and plasma phenylalanine; tyrosine andbiopterin concentrations were measured at baselineone, two, three, four, and six hours.6

Assay of dihydropteridine reductase activity. This isthe diagnostic test for inherited deficiency ofdihydropteridine reductase activity, and the assay isnow carried out on dried blood spots'4 15 in all newpatients presenting with phenylketonuria at ourclinic.These procedures were not available in our

laboratory until the end of the present study andliver biopsy was carried out in one patient formeasurement of dihydropteridine reductase andphenylalanine hydroxylase activities by Dr F Rey,Paris.5

Measurement of homovanillic acid and 5-hydroxy-indolacetic acid. The first 0*5 ml of CSF was placedon cardice at the bedside and stored at -200C.Measurements were made using high performanceliquid chromatography with electrochemicaldetection. Concentrations of homovanillic and5-hydroxyindolacetic acids in CSF provide anapproximate index of dopamine and serotoninturnover in the central nervous system'6 and havebeen used here to assess the central effects ofdisturbed biopterin metabolism. Homovanillic and

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132 Smith, Leeming, Cavanagh, and Hyland

5-hydroxyindolacetic acids in lumbar CSF are higherin early childhood than in later life,'6 so results needto be compared with controls of similar age. Onlylimited control data were available from our own

studies, but this is consistent with the data of Siefertet al. 16 Initially, these measurements were notavailable in our own laboratory, and the assays were

done by Dr M Tricklebank, London.

Results

Infants with phenylketonuria. In comparison withhealthy children6 and infants with neurologicaldisorder, plasma biopterin concentrations were

markedly raised (Fig. 2). Generally, the least risewas present in the infants with phenylalanineconcentrations closest to the normal range, but even

in the group with phenylalanine concentrationsbelow 400 [smol/I (6-6 mg/100 ml) the mean plasmabiopterin concentration (3-16 ng/ml) was signifi-cantly raised in comparison with the mean (2-2ng/ml) for infants under 2 months of age withneurological disorder (t=2-23, 0-02> p<0.05). In

22

19

E 16

.c 13-

0.

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07

4.

1*

10

0

0

l 0

0 **

0

0 t0000 *-

0

0 00 * 0 -0000o @0 4

00 0 0 0

0

°O 0

0 0 .000

0 00000

0 2

1 . . Jf1000 2000Phenylalanine (pumol/I1)

3000 36a)+

Fig. 2 Plasma biopterins measured by crithidia assayversus plasma phenylalanine concentrations in 59 infantswith phenylketonuria, pretreatment (0) and on a lowphenylalanine diet (0). - - - -=Mean value of biopterin insix control infants.Case 1 had deficiency of dihydropteridine reductase activity, and case 2 hasdefective biopterin synthesis.

Conversion: S to traditional units-Phenylalanine: 1 mmoVl- 16-5 mg/100 ml.

patients with pla,ma phenylalanine concentrationsbelow 2000 smol/l (33 mg/100 ml) there was abroadly linear relation and a significant positivecorrelation between biopterin and phenylalanineconcentrations (r=0 68, t=2-640, 0-01> p<0 02).At higher phenylalanine concentrations the scatterof results wideVied, and the linear relation was lost.The correlation between plasma biopterin andphenylalanine concentrations was closer for patientson a low phenylalanine diet (r=0-89, t=2-941,0-001> p<0-0l).

Five patients had plasma biopterin concentrationsat or outside 2SDs from the means for other patientswith similar phenylalanine concentrations (Table 1).Further investigation of biopterin metabolism inthree of the five gave normal results as follows. Inone patient with an initial biopterin concentration of18 ng/ml plasma biopterin concentration fell to thenormal range (2.1 ng/ml) when a plasma phenyl-alanine was 60 [imoUl (1 mg/100 ml), and no furtherinvestigation was undertaken. In another patientwith an initial biopterin concentration of 20*5 ng/mlthe value fell to 6-6 ng/ml when phenylalanine was700 ismoll (11-6 mg/100 ml), and an oral load oftetrahydrobiopterin produced no change in phenyl-alanine or tyrosine concentrations (Fig. 3), eventhough plasma biopterin concentrations rose duringthe test to 16 ng/ml. Dihydropteridine reductaseactivity in a dried blood spot was normal (174 [smolNADH/min/l, normal range 90-180 [tmol/l).A third patient with an initial concentration of

19-5 ng/ml was investigated elsewhere on a lowphenylalanine diet and found to have a normal urinepterin pattern. The results of tests on these threepatients indicated that the initial high biopterinvalues were due to the phenylalanine accumulationwithout deficiency of dihydropteridine reductase

Table 1 Plasma biopterins (mean (SD)) according tophenylalanine concentrations in infants withphenylketonuria due to phenylalanine hydroxylasedeficiency. Cases I and 2 omitted

No of Plasma biopterincases (nglml)

Mean (SD)

Control infants (<2 months) 6 2-20 (0-93)Phenylalanine range (ftmollt):

0-399 7 3-16 (135)400-799 23 6-54 (2-34)800-1199 7 6-87 (3-1)9)1200-1599 8 9-33 (3-40)1600-1999 7 9-8) (1t76)

62000 24 9-79 (5-09)

One way analysis of variance F=3-71, p<(-01.Conversion: SI to traditional units-Phenylalanine: I mmoVl 16-5

mg/t0( ml.

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v _ _--

_ *- - - * --

-_ / /

2 4- 16---,2 L 6 8 21.

Time (h)

Fig. 3 Plasma phenylalanine ( ) and tyrosine -- -)

concentrations after a single oral dose oftetrahydrobiopterin (BH4) 7-5 mglkg, in a patient withdihydropteridine reductase deficiency (A) and in a patientwith phenylalanine hydroxylase deficiency (a).

Conversion: SI to traditional units-Phenylalanine: 1 mmoVl= 16-5mg/100 ml.

activity. Investigations in two remaining patients,who proved to have abnormal biopterin metab-olism, are described in more detail below.

Case 1: deficiency of dihydropteridine reductaseactivity. This infant had the highest initial plasmabiopterin concentration (21-6 ng/ml) of the series.

When retested after phenylalanine concentrations

Neurological aspects of biopterin metabolism 133

had fallen to 240 ismol/l plasma biopterin concentra-tion was still high at 16-0 ng/ml. These resultssuggested a deficiency of dihydropteridine reductaseactivity, which was confirmed on further investiga-tion. Urine pterin analysis gave a biopterin: neop-terin: creatinine ratio of 814 (expected value 9-32,Dr A Niederwieser), and plasma phenylalanineconcentration fell from 344 to 99 !smol/l (5.7 to 1-6mg/100 ml) while tyrosine rose from 39 to 65 iimol/lin response to administration of tetrahydropter-idine. There was no detectable dihydropteridinereductase activity in the liver, although phenyl-alanine hydroxylase activity was normal (Dr F Rey,Paris). Concentrations of total biopterin in CSFwere raised (9-11 ng/ml) (Table 2) compared withthe results found in the patients with neurologicaldisorders (Table 3), but homovanillic (19 ng/ml) and5-hydroxyindolacetic (3.5 ng/ml) acids weremarkedly reduced and were not increased by asingle dose of tetrahydropteridine (7-5 mg/kg)(Table 2). The patient showed only minor clinicalabnormalities (hypotonia and hypomobility).

Administration of L-dopa (10-12 mg/kg), carbi-dopa (as Sinemet-110), and 5-hydroxytryptophan10-12 mg/kg from 3 months of age led to a rise in theamine metabolite concentrations in CSF (Table 2)and disappearance of the clinical signs. The patientmade good developmental progress for the next 18months, although intercurrent illness caused tran-sient hypotonia. At 10 months of age neurotrans-mitter replacement therapy was temporarilywithdrawn during a trial of monotherapy withtetrahydropteridine at a dose of 20 mg/kg/day. Afterfive days the patient became grossly hypotonic anddeveloped an acute oculogyric crisis, which wasreversed within hours of reintroduction of L-dopaand 5-hydroxytryptophan. Despite a rise of plasmatotal biopterin concentration from 11 ng/ml to 73ng/ml during administration of tetrahydropteridinethere was no rise in the already raised CSF totalbiopterin concentration. Concentrations of homo-

Table 2 Neurochemical findings in case I with deficiency of dihydropteridine reductase activity. Treatment was withL-dopa, 5-hydroxytryptophan, and carbidopa and trial was of tetrahydrobiopterin 20 mglkg/day

Pretreatment During treatment Controls (n=6)

Baseline 5 hours after L-Dopa, S-hydroxytryptophan, Tetraltydro- Rangetetrahydro- and low biopterin onlybiopterin xl phenylalanine diet 20 mg/kg

Age (months) 3-1 3.5 6 9-5 10 1-48Cerebrospinal fluid:

Homovanillic acid (ng/ml) 17 16 4(0 91 102 37 16(-605-Hydroxyindolacetic acid (ng/ml) 5 5 48 33 38 4 80-22Biopterins (ng/ml) - - - 9 11 10 3-1-44

Plasma biopterins (ng/ml) 21 27 16 _ 11 73 0-54-8

Controls=six mentally retarded children without movement disorder.

E

.5cGo.1c0 A

BH,7-5 mg /kg

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134 Smith, Leeming, Cavanagh, and Hyland

Table 3 Plasma and CSF biopterins (ng/ml) in children with neurological disorders

Group Age (yrs) Biopterin (nglml)

Mean Plasma CSF

No Mean (SD) No Mean (SD)

Mental retardation 4-2 19 1-8 (1-1) 6 3-7* (0-6)Movement disorder 4-3 22 1-7 (0-8) 11 2-3* (1-0)Torsion dystonia 12-1 5 1-7 (0-3) 4 3-6 (0-8)Inherited tics 12-1 4 2-2 (0-9)

Total 50 1-78 (0-86) 21 2-9 (0-8)

*Difference between CSF biopterin in children with mental retardation and those with movement disorder significant (t=6-22, p<0-001).

vanillic and 5-hydroxyindolacetic acids fell inaccordance with the clinical observations (Table 2).Peripheral phenylalanine accumulation was, how-ever, well controlled by a daily dose of 50 mgtetrahydrobiopterin, and this treatment wascontinued to obviate the need for a low phenyl-alanine diet. Full details of this patient have been_published elsewhere.'7

Despite good control of plasma phenylalanineconcentration and continuing amine replacementtherapy, developmental delay and regression oc-curred during the third year of life with deteriorationin speech, impaired balance, ataxia, pinpoint pupils,recurrent absences, grand mal fits, increased tone,brisk jerks, and bilateral extensor plantar responses.Low total folate concentrations in serum and CSFsuggest that the neurological deterioration was dueto interference by the accumulating biopterins withfolate metabolism,'7 and administration of folinicacid (3 mg/day) halted the neurological deteriora-tion.

Case 2: defective biopterin synthesis. A routinescreening test for phenylketonuria was negative onthe 10th day when the infant was receiving milkfeeds but had developed diarrhoea. Raised phenyl-alanine concentrations, with an excess of phenyl-ketones in the urine, was first noted at 4 months ofage after the start of complete intravenous nutritionfor persistent diarrhoea and failure to thrive. At thistime the infant weighed only 3-6 kg, had recurrentfits, was severely delayed in development, and wasnoted to be grossly hypotonic with dyskineticmovements. Plasma biopterin concentration (2-2ng/ml) was in the normal range despite plasmaphenylalanine concentrations above 2000 .tmol/l (33mg/100 ml) (Fig. 2). Withdrawal of intravenoustreatment led to a fall of phenylalanine to 600 [smol/lwithin 48 hours, but the infant suddenly collapsedand died. A dried blood spot obtained just beforedeath showed no detectable biopterin activity, and

urine analysis by high performance liquid chroma-tography showed a low total biopterin of 0-475 [tg/mland high normal neopterin of 5-325 ,tg/ml, with abiopterin:neopterin ratio of 1:11 (the normal forearly infancy being 1:2, Professor J Blair). Thesefindings suggested that the infant had a partialdefect of biopterin synthesis. Permission for postmortem was refused.

Patients with neurological disorder. The mean (SD)plasma total biopterin concentration for the 50children was 1-78 (0-86) ng/ml, almost identical tothe mean (1-75 ng/ml) found in 10 normal childrenin a previous study.6 There were no significantdifferences between groups (Table 3), and there wasno relation between age and plasma biopterin.Parents with dominantly inherited tics had similarplasma biopterin concentrations (mean 2.2 ng/ml) totheir children. Two patients, one with mentalretardation and one with movement disorder, hadinitial plasma biopterin results at the lower limit ofdetection (0-2 ng/ml). They were investigated bymeans of a phenylalanine load (Fig. 4, cases 1 and2). Fasting plasma biopterin concentrations were 1-6and 1-3 ng/ml, respectively, and the biopterinresponse to phenylalanine was normal. The lowinitial readings may have been due to artefact,perhaps caused by delay in specimen handling.CSF total biopterin concentrations were generally

higher (overall mean (SD) 2-9 (0.8) ng/ml) thanplasma values (Table 3). With the exception of fourchildren with torsion dystonia, however, patientswith movement disorder had a significantly lowermean CSF biopterin (2.3 ng/ml) than the mean forsix mentally retarded patients without movementdisorder (3-7 ng/ml, t=6.22, p<0001), and in fiveout of 11 patients with movement disorder CSFbiopterin concentration was below plasma biopterinconcentration. In five patients with torsion dystoniaCSF biopterin concentrations were similar to thosein children without movement disorder.

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3- A. .

. 2 3 4 5 6

L-phenylablcnine Time h )100 mg/kg

Fig. 4 Plasma biopterins after a load of L-phenylalanine,

100 mg/kg in three patients with neurological disorders:

-extrapyramidal disorder, 2-mental retardation, and

3-torsion dystonia.

M) and F3 mother and father of case 3. Plasma phenylalanine concentrations

rose to between 420) and 6()t 8moI/l (6-9 and 9 9 mg/l()O ml).

Converston. Sl to traditional units-Phenylalanine: ImmoVI 16 5

mg/l(10 ml.

Patient with torsion dystonia. One patient who had

normal plasma and CSF biopterin concentrations

was investigated in more detail because of a

previous report9 of reduced hydroxylase cofactor

activity in CSF in members of two families with

dystonia.

This patient presented with left intermittent

torticollis at 3 years of age. At 9 years he exhibited a

severe dystonic posture of the neck, worse in the

afternoon after eating, and disappearing during

sleep. He also had long standing swallowing diffi-

culty. He had a positive glabella tap and intermittent

torsion dystonia of the left foot. Although he was of

above average intelligence, he had great difficulty

with handwriting, during which he adopted a bizarre

posture. Neurological examination did not show any

other abnormalities. On radiological examination

there was atony and gross dilatation of the oesopha-

gus with complete absence of peristalsis and failure

of relaxation of the lower oesophageal sphincter.

There was no reflux, and the picture resembled

severe achalasia of the cardia. A fasting plasma

phenlalanine:tyrosine ratio was <1 in patient and

parents, indicating that there was no major impair-

ment of phenylalanine hydroxylation in the liver.

The response of plasma biopterin, phenylalanine,

Neurological aspects of biopterin metabolism 135

and tyrosine concentrations to a phenylalanine loadin patient and both parents (Fig. 4) was normal.Assay of dihydropteridine activity in a dried bloodspot from the patient was also normal (102 [smolNADH/min/ml). CSF homovanillic (38-5 ng/ml) and5-hydroindolacetic (8-4 ng/ml) acids seemed to below, although not as strikingly so as in the patientwith deficiency of dihydropteridine activity. Thera-peutic trials of Tegretol and of modest doses ofSinemet-110 (half a tablet thrice daily) with 5-hydroxytryptophan (20 mg thrice daily) produced noimprovement in symptoms, and the latter treatmentcaused unacceptable drowsiness. Finally, bromo-criptine in a dose of 2-5 mg twice a day abolished allsymptoms over the course of four months. Theswallowing difficulties gradually resolved, and theradiological abnormalities disappeared completely.After one year bromocriptine was withdrawn in adouble blind manner over a six month periodwithout causing clinical relapse, and six months laterthe patient remains perfectly well.

Discussion

The results reported here indicate that measurementof plasma total biopterin activity is of value in thediagnosis of patients with hyperphenylalanine due totetrahydrobiopterin deficiency. The disadvantagesof the crithidia assay are that some biopterin activityis lost in the course of the assay, it does notdistinguish the different species of biopterin, and itdoes not measure neopterins. Newer methods ofpterin analysis using high performance liquidchromatography'8 19 and radioimmunoassay2" canbe used to measure the different species of biopterinand neopterin. There will, however, be a continuingneed for simple, sensitive, and cheap methods ofbiopterin measurement for routine testing of infantswith phenylalanine accumulation. The crithidiaassay can also be applied to dried blood spots,making routine testing even simpler.'5The results of the present study, showing that

infants with raised plasma phenylalanine concentra-tions due to deficiency of phenylalanine hydroxylaseactivity have raised plasma biopterin concentrationsin proportion to the hyperphenylalaninaemia areconsistent with other studies.6 15 Among 59 con-secutive infants with persistently raised plasmaphenylalanine concentrations two patients, bothwith plasma biopterin concentrations 2SD from themean for patients with phenylalanine hydroxylasedeficiency (allowing for variation in phenylalanine),proved to have defective biopterin metabolism.The most useful investigations for confirmation or

exclusion of defective biopterin metabolism de-pended upon whether the initial plasma biopterin

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136 Smith, Leeming, Cavanagh, and Hyland

was 'high' or 'low'. In the patients with 'high' valuesmeasurement of dihydropteridine reductase activity,with repeat plasma biopterin measurement whenphenylalanine had fallen to the normal range,enabled a firm diagnosis of dihydropteridine reduc-tase deficiency to be made. In the presence of 'low'initial biopterins urine pterin analysis indicated apartial block in the conversion of neopterin tobiopterin. This patient died before an oral load oftetrahydrobiopterin could be given, and the possi-bility that biopterin deficiency was due to profoundmalnutrition, rather than to an inborn error, cannotbe excluded. The effects of disturbed biopterinmetabolism on central dopamine and serotoninsynthesis can be assessed by measurement of homo-vanillic and 5-hydroxyindolacetic acids in CSF.Although it was not performed here, it is also use-ful to measure the noradrenaline metabolite3-methoxy 4-hydroxyphenylglycol.

In the patient with dihydropteridine reductasedeficiency tetrahydrobiopterin in a single dose of 7*5mg/kg, or a daily dose of 20 mg/kg, failed to controlneurological symptoms or improve neurotransmitteramine synthesis and also failed to raise the alreadyhigh CSF total biopterin concentration. These find-ings contrast with those reported in a study of apatient with defective synthesis of biopterins inwhom it was possible to raise CSF biopterins by oraltreatment with tetrahydrobiopterin,21 and anotherstudy showing that symptoms could be controlled ina patient with a synthesis defect.'3For the first 18 months of treatment our patient

with dihydropteridine reductase deficiency madegood progress on neurotransmitter replacementtherapy, first in combination with a low phenyl-alanine diet, later with 50 mg/day tetrahydrobiop-terin. Unfortunately, as in a similar patient described

22previously, progress during the third year of lifewas less satisfactory, with evidence of neurologicaldeterioration due to a secondary defect of folatemetabolism. 17

In 50 children with neurological disease andwithout hyperphenylalaninaemia plasma biopterinconcentrations were similar to those of healthychildren,6 suggesting that in patients with normalphenylalanine concentrations general disturbance ofbiopterin metabolism is rarely the cause of progres-sive intellectual loss or extrapyramidal disturbance.CSF biopterin concentrations in children withoutmovement disorder and children with torsion dysto-nia were similar to those in children with leukaemia(4-2 ng/ml) studied before treatment (unpublisheddata). CSF biopterin concentrations were lower inchildren with movement disorder other than torsiondystonia, a finding which can best be explained asdue to anatomical or functional abnormality of the

brain areas normally contributing to CSF biopterinrather than as the result of a primary defect ofbiopterin metabolism.

Six adult patients with Huntington's chorea alsohad normal plasma biopterin and low CSF totalbiopterin concentrations (1-9 ng/ml) (unpublishedobservations). A study of 'hydroxylase cofactoractivity' in CSF found concentrations equivalent to20 iimol/l tetrahydrobiopterin (4.2 ng/ml) in adultcontrols, and patients with movement disorder(Parkinson's disease and Huntington's chorea) againshowed a reduction of 50% or more.8

Profoundly low CSF 'hydroxylase cofactor activ-ity' has been reported in several members of twofamilies with torsion dystonia.9 These patients didnot manifest the degree of neurological disturbancethat might have been expected2 if tetrahydrobiop-terin in the central nervous system had indeed beenseverely reduced. These results contrast with thenormal crithidia results in the patients with torsiondystonia described here, although one in whomamine metabolite concentrations were measured didshow reduced concentrations of dopamine andserotonin metabolites in the CSF, suggesting thatinterference with tyrosine and tryptophan hydroxy-lation might have been present. We speculate thatan inhibitor of the aromatic hydroxylases is presentin the CSF and brains of some patients with torsiondystonia, which, without direct interference inbiopterin synthesis, reduces 'hydroxylase cofactoractivity' and amine turnover. The association re-ported here of achalasia of the cardia with torsiondystonia and a disturbance of amine metabolism isof interest as the pathogenesis of both disordersremains a mystery.

We thank Dr M Tricklebank, Dr A Niederwieser, Dr FrancoiseRey, and Professor J A Blair for their generous help with theinvestigations, Dr E Brett, Dr J Wilson, and Professor 0 H Wolfffor allowing us to investigate their patients, Dr T Crow for donatingthe CSF from patients with Huntington's chorea, and Professor GCurzon for many helpful discussions. Financial support for thiswork was provided by the McAlpine Foundation and the MedicalResearch Council.

References

Kaufman S. Regulatory properties of pterin dependent hyd-roxylases. Variations on a theme. In: Usdin E, Weiner N,Youdim MBH, eds. Function and regulation of monoamineenzymes. McMillan Publications Ltd, 1981:165-73.

2 Smith I, Clayton BE, Wolff OH. New variant of phenylketo-nuria with progressive neurological illness unresponsive tophenylalanine restriction. Lancet 1975;i:1108-11.

3 Danks DM, Bartholome K, Clayton BE, et al. Malignanthyperphenylalaninaemia-current status. Journal of InheritedMetabolic Disease 1977;1:49-53.Kaufman S, Holtzman NA, Milstien S, Butler IJ, Krumholz A.Phenylketonuria due to a deficiency of dihydropteridine reduc-tase. N Engl J Med 1975;293:785-90.

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Neurological aspects of biopterin metabolism 137

5 Rey F, Harpey JP, Leeming RJ, Blair JA, Aicardi J, Rey J.Les hyperphenylalaninemies avec activite normale de laphenylalanine-hydroxylase. Le deficit en tetrahydrobiopterineet le deficit en dihydropteridine reductase. Arch Fr Pediatr1977;34(suppl II): 109-120.

6 Leeming RJ, Blair JA, Green A, Raine DN. Biopterinderivatives in normal and phenylketonuric patients after oralloads of L-phenylalanine, L-tyrosine and L-tryptophan. ArchDis Child 1976;51:771-7.

7 Rey F, Saudubray JM, Leeming RJ, Niederwieser A, CurtiusH-Ch, Rey J. Les deficits partiels en tetrahydrobiopterine. ArchFr Pediatr 1983;40(suppl I):237-41.Williams AC, Levine RA, Chase TN, Lovenberg W, Calne DB.CSF hydroxylase cofactor levels in some neurological diseases.J Neurol Neurosurg Psychiatry 1980;43:735-8.

9 Williams A, Eldridge R, Levine R, Lovenberg W, Paulson G.Low CSF hydroxylase cofactor (tetrahydrobiopterin) levels ininherited dystonia. Lancet 1979;ii:410-11.

1" Barford PA, Blair JA, Eggar C, Hamon C, Morar C, WhitburnSB. Tetrahydrobiopterin metabolism in the temporal lobe ofpatients dying with senile dementia of Alzheimer type. J NeurolNeurosurg Psychiatry 1984;47:136-8.

" Leeming RJ, Blair JA. The effects of pathological and normalphysiological processes on biopterin derivative levels in man.Clin Chim Acda 1980;108:103-11.

12 Niederwieser A, Curtius H-Ch, Gitzelmann R, et al. Excretionof pterins in phenylketonuria and phenylketonuria variants.Helv Paediatr Acta 1980;35:335-42.

13 Niederwieser A, Curtius H-Ch, Wang M, Leupold D. Atypicalphenylketonuria with defective biopterin metabolism.Monotherapy with tetrahydrobiopterin or sepiapterin. Screen-ing and study of biosynthesis in man. Eur J Pediatr1982;138:1 10-2.

14 Arai N, Narisawa K, Hagakawa H, Tada K. Hyperphenylalani-

naemia due to dihydropteridine reductase deficiency: diagnosisof enzyme assays on dried blood spots. Pediatrics 1982;70:426-30.

5' Leeming RJ, Barford PA, Blair JA, Smith I. Blood spots onGuthrie cards can be used for inherited tetrahydrobiopterindeficiency screening in hyperphenylalaninaemic infants. ArchDis Child 1984;59:58-61.

16 Siefert WE, Fox JL, Butler IJ. Age effect on dopamine andserotonin metabolite levels in cerebrospinal fluid. Ann Neurol1980;8:38-42.

17 Smith I, Hyland K, Kendall B, Leeming R. Clinical role ofpteridine therapy in tetrahydrobiopterin deficiency. Journal ofInherited Metabolic Disease 1985;8(Suppl 1):39-45.

18 Fukushima M, Nixon JC. Analysis of reduced forms of biopterinin biological tissues and fluids. Anal Biochem 1980;102:176-88.

'9 Lunte CE, Kissinger PT. Determination of pterins in biologicalsamples by liquid chromatography/electrochemistry with dualelectrode detector. Anal Chem 1983;55:1462-6.

2(0 Nagatsu T, Yamaguchi T, Kato T. Biopterin in human brain andurine from controls and Parkinsonian patients: application of anew radio-immuno assay. Clin Chim Acta 1981;109:305-11.

21 Kaufman S, Kapatos G, McInnes RR, Schulman JD, Rizzo WB.Use of tetrahydrobiopterins in the treatment of hyperphenyl-alaninaemia due to defective synthesis of tetrahydrobiopterin:evidence that peripherally administered tetrahydropterins enterthe brain. Pediatrics 1982;70:376-80.

22 Harpey JP. Les defauts de synthese des biopterines: les deficitscomplets (reductase et synthetase). Arch Fr Pediatr 1983;40:231-5.

Correspondence to Dr I Smith, Institute of Child Health, 30Guilford Street, London WC1N 1EH.

Received 18 October 1985

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