Citrullinemia: Phenotypic Variations

(1)

Citrullinemia:

Phenotypic

Variations

Donald T. Whelan, M.D., Terry Brusso, B.H.Sc., R.P.Dt.,

and Marilyn Spate, B.A.

Front the Departments of Pediatrics, Pathology, and Nutritional Services, Mc’tfaster Unicersity Medical

Centre, Hamilton, Ontario, Canada

ABSTRACT. An 18-month-old female infant was found to

have citrullinemia on routine plasma screening by the

Scriver Method at 5 days of age. At 10 days of age, plasma citrulline concentration was 0.7O4jzmol/mI (normal, 0.010 to 0.O3Otmol/ml) and has remained 60 to 80 times higher than normal. Urine citrulline concentration was markedly ele-vated. Hyperammonemia occurred at 1 month of age. The senim ammonia concentration was 473tg/100 ml (normal, 50 to 2SOjig/ 100 ml) and rose to 770tg/ 100 ml at 4 months of age. Dietary protein was restricted to 1.6 gm/kg/day. Without further change in protein intake, the serum ammonia concentration decreased to 280.sg/100 ml and, since then, it has returned to normal. The addition of three synthetic L-amino acids was required for a short time during dietary therapy. At 10 months of age, the infant was given a normal diet. At 18 months of age, her physical and mental development is normal. Activity of argininosuccinic acid synthetase measured in skin fibroblasts was 0.0037smo1 of radioactive carbon dioxide per milligram of protein per hour. To demonstrate heterozygosity, fasting plasma citrulline concentrations were measured in five members of the family.

Comparison of findings in this patient with those reported in the literature suggests pbenotpical variation of the disease, probably due to genetic heterogeneity. Pediatrics, 57:936-94 1, 1976, CITRULLINEM IA, GENETICS, PHENOTYPES,

PLASMA SCREENING.

Disorders

of

the

Krebs-Henseleit

urea

cycle

were

first

described

by

Allan

et a!.1

and

Levin

et al.2 They documented clinical and biochemical

data

on

the

disorder

known

as

argininosuccinic

aciduria.

Inborn

errors

of

all

of

the

metabol-ic reactions in that cycle have now been

de-scribed.

Citrullinemia

was

first

described

by

McMurray

et

al.3

Since then, 12 additional

patients

have

been

reported

in the

literature.

The

disorder

is caused

by

deficient

activity

of

argini-nosuccinic

acid

synthetase

(E.C.6.3.4.5).

This

results

in

a marked

elevation

in

the

concentra-tions

of plasma

citrulline

and

ammonia.

A lack

of

the

ultimate

product

of

this

pathway

should

occur,

but

does

not.

Alternate

pathways

for

the

production of urea have been investigated.

Clini-cally

the

picture

varies,

ranging

from

a

devas-tating

metabolic

derangement

presenting

with

vomiting,

convulsions,

coma,

and

death

to

an

apparently

benign

biochemical

anomaly

with

no

clinical

manifestations.

Treatment

consists

of

a

low-protein

diet,

but

has

not

been

highly

success-ful.

Citrullinemia

is

likely

to

be

genetically

and

phenotypically

heterogeneous,

as are

many

other

such

diseases.5

This

heterogeneity

has

important

connotations

regarding

detection,

treatment,

and

prognosis.

CASE REPORT

K.U. is the firstborn female child of unrelated parents. On

the fifth day of life, routine plasma amino acid screening detected an elevation of citrulline. A urine sample also showed a marked increase in citrulline. Physical examination

at 9 days of age revealed an asymptomatic child in no acute

(Received August 12; revision accepted for publication October 3, 1975.)

Supported in part by FDA contract 72-304.

ADDRESS FOR REPRINTS: (D.T.W.) Room 2N26, McMas-ter University Medical Centre, 1200 Main Street West,

(2)

TABLE I

BIOCHEMICAL DATA ON PATIENT K. U. AT VARIOUS AGES

Age

Urine Citrulline

(pg/gm of creatinine)#{176}

Serum Ammonia

(mg/100 ml)t

BUN

(mg/100 ml)

Dietary Protein

(gm/kg)

Weight

(kg) Remarks

9 days - 64 7.5 1.70 - Skin biopsy; EEG normal; protein

restriction initiated

33 days - 473 7.0 1.30 3.90

50 days 1,073.0 500 8.0 1.30 4.10 First immunization

4 mo - 770 8.0 1.65

-5 mo 1,706.0 225 5.0 1.70 5.44 Dietary addition of

L-phenylalan-me, L-isoleucine, and L-leucine; start dicalcium phosphate and vitamin D

7 mo 1,181.0 200 - 1.80

-10.5 mo 4,489.5 152 18.0 2.60 7.75 Changed from SMA to 2% milk

with a marked increase in pro-tein intake at 8.5 mo

14 mo 1,640.5 29 - - - Upper respiratory tract infection

18 mo 914.6 45 11.0 4.2 9.90 Normal mental and physical

devel-opment

‘Normal = < 10 mg/gIn of creatinine. tNormal, 0 to 200tg/100 ml.

distress and with no abnormal physical findings. She was admitted to hospital for further investigation. Plasma citrul-line was 0.704mo1/ml. Blood urea nitrogen (BUN) was 7.5 mg/100 ml. Serum ammonia was 64g/100 ml. EEC was

normal. Because of the normal serum ammonia, therapy was

not initiated. A skin biopsy was obtained from the forearm in order to obtain cultured fibroblasts for enzyme assay. She

was followed very closely, and two weeks later her clinical biochemical status had not changed, except that her serum ammonia had risen to 473sg/ 100 ml (Table I). Calculated protein intake was 1.7 gm/kg/day. A decision was made to lower the dietary protein to levels which would still sustain normal growth. Breast milk was still available and the mother initiated breast-feeding. Tri-Vi-Sol vitamin prepara-tion was added to the diet. The daily protein was now

approximately 1.3 gm/kg/day. During the first two months,

the concentration of citrulline in the plasma and urine of this

patient remained extremely elevated. Her BUN was normal.

Her serum ammonia remained elevated. Her growth rate started to decrease and therefore her daily protein intake was gradually increased to approximately 1.7 gm/kg/day. There was a marked acceleration in growth with no change in her

plasma citrulline concentration. Her serum ammonia rose to

770tg/ 100 ml. At this time, breast-feeding was discontinued and formula was begun so that more accurate dietary protein calculations could be obtained. Subsequent to this, her serum ammonia gradually returned to within normal limits. Her dietary protein intake was slowly increased and there was no change in her clinical or biochemical status. At 5#{189}months of age, a nutritional survey and plasma amino acid

concen-tration determinations indicated that the minimum daily

requirements for three of the essential amino acids had

not been met. Therefore, 100 mg/day of L-isoleucine, DL-phenylalanine and L-leucine were added to the diet. There was another rapid acceleration of growth. For the next

4#{189}months, her plasma and urinary citrulline remained

elevated and her serum ammonia remained at the upper limit of normal. At 8#{189}months of age, since the serum ammonia was not increasing, and the plasma citrulline

seemed stable, we switched the infant to a normal diet which included 2% cow’s milk. The protein intake was now 2.6 gm/ kg/day. From that time to 18 months of age, physical and mental development have proceeded normally, yet her plasma and urinary citrulline remain markedly elevated. Her

senuTi ammonia has remained within normal limits.

MATERIALS AND METHODS

Plasma amino acids were identified

semiquan-titatively

utilizing

unidimensional

paper

chroma-tography.1 Plasma citrulline was identified by

overstaining

the

chromatogram

with

Ehrlichs

II

Urine

amino

acids

were

identified

semi-quantitatively

using

two-dimensional

paper

chro-1 2 Urinary citrulline was also

identi-fled

by overstaining

with

Ehrlichs

reagent.

Quan-titation

of amino

acids

was

obtained

by

elution

chromatography” on a Beckman Amino Acid Analyser 120C. The analyser utilizes an ion

ex-change

resin

(UR-30)

and

elution

is with

lithium

buffers. The Ninhydrin and Ehrlichs positive

compound

in plasma

and

urine

did

co-chromato-graph

with

a

known

standard

of

L-citrulline.

Orotic acid concentration was determined on

random

urine

samples

at various

times

during

the

first 18 months of age. The method measured

(3)

I

GLUTAMIC ACID

I

HCO32ATP

IC4!i

__

CARBAMYL PHOSPHATE - PYRIMIDINE SYNTHESIS

0 R N

ITHJI1I1S

CITRULLINE (P1)

ATP ASPARTIC ACID

ARGINASE

AR G IN IN E

FU MAR IC ACID

I

ASA

SYNTHAJ

I

ARGININOSUCCINATE (AMP + PP)

REGULATION

OF

BLOOD

AMMONIA

CITRIC ACID CYCLE

D EAM INATION

OF AMINO ACIDS

I

1

- KETOGLUTARATE

F

1NH

GLUTAMINE

SYNTHETASE

GLUTAMINE

GLUTAMINASE

1 ARGININOSUCCINASE]

FIG. 1. The three major biochemical pathways utilized in the metabolism of ammonia.

TABLE II

PLASMA AMINo ACID LEVELS OF PATIENT K. U. AT Vusious AGES

Amino Acid

Normal Range’

9 Days

33 Days

50 Days

4 Months

5 Months

7 Months

10 Months

14 Months

18 AIonths

L-Citrulline 0.012 to 0.030 0.704 0.704 0.470 0.460 0.478 0.840 0.643 0.736 0.583

L-Lysine 0.071 to 0.151 0.102 0.282 0.174 0.026 0.066 0.061 0.200 0.173 0.40

L-Proline 0.068 to 0.148 0.188 0.054 0.069 0.620 0.416 - 0.134 0.484 0.091

L-Arginine 0.023 to 0.086 0.039 0.099 0.058 0.032 0.049 0.032 0.038 0.051 0.023

L-Ornithine 0.027 to 0.086 0.026 0.119 0.164 0.044 0.042 0.165 0.055 0.056 0.017

L-Glycine 0.117 to 0.223 0.186 0.156 0.177 0.080 0.154 0.154 0.115 0.405 0.232

L-Alanine 0.137 to 0.305 0.308 0.311 0.325 0.127 0.364 0.447 0.306 0.555 0.385

L-Glutamine 0.057 to 0.467 - - - - 0.384 0.665 0.616 0.690 0.69()

Glutamic acid 0.023 to 0.250 0.029 - - 0.191 0.077 0.074 0.066 0.88 0.055

L-Phenylalanine 0.026 to 0.061 0.047 0.053 0.034 0.030 0.030 0.069 0.062 0.062 0.046

L-Leucine 0.056 to 0.178 0.136 0.170 0.066 0.051 0.053 0.130 0.143 0.1 12 0.()92

L-Isoleucine 0.028 to 0.084 0.053 0.071 0.020 0.037 0.043 0.095 0.078 0.069 0.047

Aspartic acid 0.004 to 0.020 0.034 - - 0.014 0.012 0.026 0.014 0.007 0.012

(4)

TABLE III

FASTING PLASMA CITRULLINE VALUES FOR PATIENT K. U’s

FAMILY

0

Relative Gitrulline (tmol/ml)

Mother 0.0441

Father 0.0748

Maternal grandfather 0.0365

Maternal grandmother 0.079 Paternal grandmother 0.0559 Control

Range 0.006 to 0.033

Mean 0.019

using the optical density readings at 480jt and

412t

to obtain

a ratio

as described

by

Rogers

and

Porter.

To determine serum ammonia, venous blood

samples

were

obtained

under

similar

conditions

at

each clinic visit (1 1:30 AM). The antecubital vein

was

used

and

no

tourniquet

was

applied.

The

blood

sample

was

drawn

into

a

plastic

3-ml

disposable

syringe

and

immediately

transferred

to

a

cold

3-ml

stoppered

glass

tube

on

ice.

The

ammonia

determination

was

done

within

15 minutes.

The

method

uses

the

ESKALAB

am-monia

reagent

available

from

Smith-Kline

Instru-ments.

The

method

used

for

BUN

determination

was

a

modification

of that

described

by

Marsh

et al.15

A skin biopsy taken from the forearm was

placed

in

Eagle’s

minimum

essential

media,

supplemented

with

sodium

pyruvate,

ferric

nitrate,

and

15%

fetal

calf

serum.

Cells

were

cultured

in a moisturized

incubator

at 37 C in an

atmosphere at 5% carbon dioxide in air.

Arginino-succinic

acid

synthetase

activity

was

measured

using.

the

modified

radiochemical

procedure

described

by

Schimke.’

Fasting

venous

blood

samples

were

taken

from

five

members

of the

family

for studies

of citrulline

metabolism in an attempt to identify heterozy-gotes. Quantitation of plasma amino acids were

obtained

using

the

techniques

described

above.

RESULTS

Plasma Amino Acids

Plasma

amino

acid

concentrations

in

the

pa-tient

are

shown

in

Table

II. L-citrulline,

L-alanine, and L-glutamine were consistently

elevated.

L-arginine

was

usually

found

to be

low.

The

plasma

concentration

of

L-lysine

and

L-glutamic

acid

did

not

show

any

major

fluctua-tions

from

normal.

The

quantitation

of

homoci-trulline

and

homoarginine

was

attempted,

and

the

presence

of

these

amino

acids

was

never

detectable.

Urine Amino Acids

Consistent

urinary

quantitation

of amino

acids

was not possible, but urinary citrulline was always markedly elevated. Homocitrulline and

homoar-ginine

were

looked

for,

but

we

were

never

able

to

detect

the

presence

of

these

amino

acids

in

urine.

Urine Orotic Acid

The

excretion

of orotic

acid

was

measured

on

numerous

occasions.

No

significant

changes

were

seen

when

dietary

protein

was

changed.

On

ten

different

occasions

during

the

first

18 months

of

life,

the

ratio

of the

optical

density

at

48Oi

and

412jt

gave

a mean

reading

of 0.53

(range,

0.290 to

0.767).

The

normal

ratio

is less

than

0.5.

BUN

This

measurement

was

always

within

normal

limits.

The

values

ranged

from

5.0 to 18.0

gm/100

ml.

Increased

BUN

values

correlated

with

the

increase

in dietary

protein.

Serum Ammonia

A normal value of 64tg/ 100 ml. was reported

at 9 days

of age.

At one

month

of age,

there

was

a

sudden

increase

in

ammonia

concentration

to

473ig/100 ml; it remained elevated for 3 to 4

months,

then

gradually

returned

to normal.

At

18 months

of age,

the

serum

ammonia

was

45tg/

100 ml.

Family Studies

Fasting

plasma

citrulline

values

from

five

members

of

the

patient’s

family

are

shown

in

Table

III. The

mother,

father,

and

both

grand-mothers

have

citrulline

concentrations

greater

than

normal

controls.

Argininosuccinic

Acid Synthetase

Activity

The

specific

activity

of

argininosuccinic

acid

synthetase in cultured skin fibroblasts was

0.0037tmol

of

radioactive

carbon

dioxide

per

milligram

of

protein

per

hour.

Age-matched

control

cells

assayed

at

the

same

time,

gave

a

value

of 0.079tmol/mg

of protein

per

hour.

This

represents

a reduction

to 5% of normal

activity

in

the

patient.

DISCUSSION

(5)

hyperani-TABLE IV

PHENOTYPIC VARIATIONS IN PATIENTS WITH CITRULLINEMIA

Age at Author Onset Clinical Sigiu and Symptoms Plasma Citrulline (jimol/mi)’ Serum Ammonia (pg/100 ml) Enzyme Activity (% control) BUN

(mg/ 100 ml) Outcome

Wick et al.2 3 days Hypertonia,

poor feed-ing, coma

3.10 ? Liver 20%,

brain 30%

16.7 Death at 6 days

Wick et al.2’ 2 days Hypertonia,

convulsions

2.20 2,550 Liver 14%,

brain 17%

15.0 Death at 3 days

Ghisolfi et 12 hr al.’

Hypertonia 2.20 170 Not done - Death at 6 days

VanDerZee 4 days et al.’

Hypotonia, convul-sionS, coma

4.57 P Liver 0%,

brain 0%

Normal Death at 7 days

Roerdink et 3 days al.’2 Drowsiness, convulsions 2.75 Normal, 77 Liver 0%, brain normal

4.0 Death at 4 days

Danks et al. 1 day Irritability, family his-tory

0.80 154 - 15.0 Treated: death at 7.5 mo

with infection

Vidailhet et 2 mo al.3’

Vomiting,

slow devel-opment

2.20 1,300 Liver 0%,

kidney normal

- Treated: death at 7.5 mo with infection

McMurray et 9 mo al.

Vomiting, hpotonia

1.50 1,000 Liver 5% Normal Mentally retarded

Scott-Emuak- 12 mo por et al.2 ‘

Vomiting, con-vulsions

1.47 155 Skin 1% - Mentally retarded at 33 yr

Buist et al. 2 mo Semicoma,

hepato-megaly

4.50 212 Skin 1% 7.0 Normal at 4 yr

Wick et al.2 3 wk None 0.315 170 Skin 75% - Normal at 3 yr

Miyazaki et 21 yr al.7

Slurred speech

0.882 350 - - Normal

Whelan et a!. - None 0.824 770 to 29 Skin 5% 8.0 Physically and mentally

normal at 18 mo

‘Normal, 0.012 to 0.030 .tmol/m1.

monemia,

elevation

of the

immediate

or

remote

substrates, and a deficiency of the products.’

There

are

other

inborn

errors

of

amino

acid

metabolism which also result in

hyperammone-mia.’TM Citrulline is an intermediate substance in

the

cyclic

mechanism

resulting

in the

production

of

urea

from

ammonia,

carbon

dioxide,

and

aspartic acid. A block in the pathway distal to citrulline should lead to an elevation of citrulline

and

ammonia

along

with

a deficiency

of arginine

and

urea.

Which

of these

substrates

is responsible

for the morbidity and mortality associated with this disorder is questionable, but ammonia is suspect. Okken

et

al.”

suggest that elevated citrulline concentrations can be toxic to a basic

metabolic

process

in

the

brain.

All

the

patients

reported

to date,

including

the

one

reported

here,

have had marked elevation of plasma citrulline ranging from 0.315 to 4.57 jtmol/ml (normal,

0.001

to 0.030imol/ml)

as shown

in Table

IV. The

first

mechanism

regulating

ammonia

metabolism

involves

the

reversible

conversion

of

a-ketoglutarate to glutamic acid and glutamine, which will provide ammonia for renal excretion. (Fig. 1). The second mechanism is the synthesis of

pyrimidmes for nucleic acid synthesis via

carba-mylaspartate.

The

third

and

major

function

of the

Krebs-Henseleit urea cycle is the excretion of the

nitrogen unnecessary for body needs as urea. The

abnormalities seen in the concentrations of plasma alanine and glutamine reflect a

wide-spread

distortion

of

ammonia

metabolism,

per-haps indicating the functioning of the first

mech-anism alluded to above. The normal urinary

orotic acid excretion found in our patient

mdi-cates

that

the

second

mechanism

was

not

func-tioning

at

a greater

capacity

than

normal.

The

observation of normal urea levels in the blood of

our patient demonstrates that the urea cycle is

(6)

unable

to

produce

urea

has

ever

survived,

suggesting

that

a complete

block

in

the

produc-tion

of urea

is incompatible

with

life.

The

initial

rise

in the

serum

ammonia

and

the

gradual decrease back to normal concentrations suggest that perhaps an alternative mechanism

for

the

metabolism

of ammonia

and

the

synthesis

of urea

is functioning

in our

patient.

Tedesco

and

Mellman2

demonstrated

an

elevated

Michaelis

constant

(Km)

of argininosuccinic

acid

synthetase

for its substrate. Kinetic studies were not

per-formed

in the

skin

fibroblasts

of our

patient.

The

alternate pathway for the synthesis of urea,

described

by

Scott-Emuakpor

et al.,2’

is unlikely

to be

operative

in our

patient,

since

no

homoci-tnilline

or

homoarginine

could

be

detected

in

urine

or plasma.

Levin

et al.22

rejected

the

possi-bility

of

a late-developing

alternate

urea

cycle

and

postulated

that

a competitive

inhibition

of

lysine metabolism by citrulline exists. This patient

did

not

demonstrate

elevated

concentrations

of

L-lysine and, therefore, this mechanism was not

given

much

significance.

Cathelineau

et al.2’

offered

yet

a third

explana-tion

for

the

amino

acid

findings

reported

by

the

above

investigators,

which

needs

to be considered

in these

patients.

They

postulate

that

L-lysine

and

L-homocitrislline

accumulate

because

they

are

substrates

of ornithine

transcarbamylase

and

argi-ninosuccinic acid synthetase respectively. Cohen

et al.2’

have

postulated

the

existence

of a minor

pathway

for

urea

synthesis

that

utilizes

guanidi-nosuccinic

acid

as an

intermediate.

Stein

et al.25

concluded

that

an

intact

ornithine-urea

pathway

is necessary for such a mechanism to function.

Therefore,

we did

not

search

for

either

guanidino-succinic acid or guanidinoacetic acid. Our

patient’s

plasma

and

urine

creatinine

were

normal

each

time

they

were

determined.

A review

of

the

clinical

and

biochemical

pa-rameters in the recorded cases of citrullinemia indicate

that

significant

phenotypical

variation

exists.

This

heterogeneity

should

be

entertained

when

considering

the

prognosis

of

each

case.

Table

IV shows

the

various

clinical

and

biochem-ical

parameters

by

which

patients

with

citrulli-nemia

can

be

divided

into

three

distinct

groups.

We

are

aware

that

oral

citrulline

tests

do

not

discriminate

between

carriers

and

controls.

We

did

not

attempt

to obtain

skin

biopsies

from

the

family to assay argininosuccinic acid synthetase as suggested by Buist et al.26 Elevated fasting

plas-ma

citrulline levels have been reported in carriers

of

citnillinemia.2

Our

results

show

the

fasting

plasma citrulline levels elevated in four of five

family

members

tested.

On

this

evidence,

they

were considered to be probable heterozygotes.

Because

the

patient

reported

here

was

detected

so early

in life,

and

prior

to any

clinical

manifes-tations,

it is difficult

to determine

to which

group

of patients

she

belongs.

Because

she

was

asymp-tomatic,

and

her

plasma

citrulline

values

have

never gone higher than 0.840tmol/ml she

probably

would

not

fit into

group

one.

We

were

more

concerned

with

the

effects

of

hyperammo-nemia

than

hypercitrullinemia

and/or

hypoargi-ninemia

and

initiated

therapy

only

when

the

former existed. It seems that the dietary

restric-tion

of

protein

in

this

case

had

little

or

no

immediate

effect

on

the

plasma

citrulline

con-centration

or

the

serum

concentration

of

ammo-nia.

However,

we

did

observe

a gradual

decrease

in plasma

citrulline

concentration

to 0.470jtmol/

ml.

At

2 months

of age,

her

growth

rate

slowed

and

it wa

necessary to increase her protein intake

to

1.65 gm/kg/day.

Her

plasma

citrulline

concentration

returned

to

the

high

values.

Because

of this,

we

did

contemplate

other

forms

of

therapy.28

Hypoargininemia

was

never

detected

in our

patient.

There

was

a spontaneous

and

gradual

return

of

the

serum

ammonia

to

normal.

The

possible

mechanisms

responsible

for

this

have

been

discussed.

Enzyme

activity

in

cultured skin fibroblasts demonstrated a 5%

residual

activity.

This

patient

demonstrates

that

infants

can

survive

and

develop

normally

with

persistently high concentrations of plasma

citrul-line.

SUMMARY

The

infant

reported

in this

paper

was

detected

at an

early

age,

and

before

any

clinical

signs

or

symptoms

became

manifest.

She

has

had

persis-tent

citrullinemia

and

transient

hyperammone-mia

which

subsided

spontaneously.

The

various

mechanisms

which

could

account

for

these

biochemical

findings

are,

discussed.

Her

enzyme

activity

in cultured

skin

fibroblasts

showed

a 5%

residual

activity.

Therapy

was

initiated

for

the

hyperammonemia,

but

had

no effect

on ammonia

or citrulline

concentration.

Fasting

plasma

citrul-line

concentrations

were

obtained

on

family

members

and

four

out

of five

were

elevated

above

the

normal

range

for

this

amino

acid.

A review

of the

patients

who

have

citrullinemia

previously

investigated

show

that

there

are

three

distinct

groups

of patients.

Citrullinemia,

one

of

the

inborn

errors

of

the

Krebs-Henseleit

urea

cycle, does show genetic heterogeneity by dem-onstrating phenotypic variations in its

(7)

REFERENCES

1. Allan JD, Cusworth DC, Dent CE, Wilson UK: A disease, probably hereditary, characterized by severe mental deficiency and a constant gross abnormality of amino acid metabolism. Lancet 1:182, 1958.

2. Levin B, McKay HMM, Oberholzer VG: Argininosuc-cinic acid, an inborn error of amino acid metaho-lism. Arch Dis Child 36:622, 1961.

3. Hommes FA, DeGroot CJ, Wilmink CW, Janxis JHP: Carbamylphosphate synthetase deficiency in an infant with severe cerebral damage. Arch Dis Child 44:688, 1969.

4. Short EM, Conn HO, Snodgrass PJ, et a!: Evidence for X-linked dominant inheritance of ornithine trans-carbamylase deficiency. N Engl

J

Med 288:7, 1973.

5. McMurray \VC, Mohyuddin F, Bayer SM, Rathhun JC: Citrullinemia, a disorder of amino acid metabolism associated with mental retardation. In, Oster

J,

et a!

(

eds): Proceedings of the International Congress on the Scientific Study of Mental Retardation. Copen-hagen, 1964, p 117.

6. Hambraeuis L, Hardell LI, Westphol 0, et a!: Arginino-succinic aciduria. Acta Paediatr Scand 63:525, 1974.

7. Terheggen HG, Lavinha F, Colombo JP, et a!: Familial hyperargininemia.

J

Genet Hum 20:69, 1974. 8. Child.s B, DerKaloustian VM: Genetic heterogeneity. N

Engi

J

Med 279:1205, 1968.

9. Holt LE, Snyderinan SE: Amino acid requirements of infants. JAMA 75:100, 1961.

10. Scriver CR, Davis E, Cullen AM: Application of a simple micromethod to the screening of plasma for a variety of aminoacidopathies. Lancet 2:230, 1964.

11. Smith I (ed): Chromatographic and Electrophoretic Techniques. New York, William Heinemann, 1969, p 253.

12. Dent CE: Study of behavior of some sixty amino acids and other ninhydrin-reacting substances of phenol-collidine filter paper chromatograms, with notes as to occurrence of some of them in biological fluids. Biochem

J

43:169, 1948.

13. Moore 5, Spackman DH, Stem WH: Chromatography of amino acids on sulfonated polystyrene resins: An improved system. Anal Chem 30:1185, 1958. 14. Rogers LE, Porter FD: Hereditary orotic aciduria: A

urinalv screening test. Pediatrics 42:423, 1968. 15. Marsh WH, Fingerbut B, Miller H: Automated and

manual direct methods for the determination of l)loOd urea. Clin Chem 11:624, 1965.

16. Schimke RT: Enzymes of arginine metabolism in mammalian cell culture.

J

Biol Chem 239:136, 1964.

17. Scriver CR: Treatment in medical genetics. In, Crow JE, Ned JV (eds): Proceedings of the Third Interna-tional Congress of Human Genetics. Baltimore, Johns Hopkins Press, 1967, pp 45-56.

18. Hsia EY: Inherited hyperammonemic syndromes. Gastroenterology 67:347, 1974.

19. Okken A, VanderBliz JF, Hommes FA: Citrullinemia and brain damage. Pediatr Res 7:52, 1973.

20. Tedesco TA, Mellman WJ: Argininosuccinate synthe-tase activity and citrulline metabolism in cells cultures from a citrullinemic subject. Proc Natl

Acad Sci 57:829, 1967.

21. Scott-Emuakpor A, Higgins JV, Kohrman AF:

Citrul-linemia: A new case with implications concerning

adaptation to defective urea synthesis. Pediatr Res 6:626, 1972.

22. Levin B, Oberholzer VG, Palmer T: Citrullinemia and an alternative urea cycle, letter. Pediatr Res 7:728, 1973.

23. Cathelineau L, Saudubray JM, Charpetier C, Polonovski

C: Letter. Pediatr Res 8:857, 1974.

24. Cohen BD, Stein IM, Bonas JE: Guanidinosuccinic aciduria in uremia: A possible alternate pathway for urea synthesis. Am

J

Med 45:63, 1968.

25. Stein IM, Cohen BD, Kornhauser RS: Guanidinosuccinic acid in renal failure, experimental ajotemia and inborn errors of the urea cycle. N Engl

J

Med

280:926, 1969.

26. Buist NRM, Kennaway NC, Hepburn CA, et a!: Citrul-linemia: Investigation and treatment over a four year period.

J

Pediatr 85:208, 1974.

27. Wick H, Bruchbiihler T, Gerard

J:

Citrullinemia: Elevated serum citrulline levels in healthy siblings. Experientia 26:823, 1970.

28. Levin B, Russell A: Treatment of hyperammonemia. Am

J

Dis Child 113:142, 1967.

29. Wick H, Bachmann C, Baumgartner R, et a!: Variants of

citrullinemia. Arch Dis Child 48:636, 1973. 30. Ghisolfi

J,

Augies D, Martinez

J,

et a!: Forme neo-natale

de citrullinmeic a evolution martelle rapide. Pedi-atrie 27:55, 1972.

31. VanDerZee SPM, Trijbels JMF, Morinens LAH, et a!: Citrullinemia with rapidly fatal neonatal course. Arch Dis Child 46:847, 1971.

32. Roerdink FH, Gouw WLM: Citrullinemia: Report of a case with studies on ant.enatal diagnosis. Pediatr Res 7:863, 1973.

33. Danks DM, Tippett P, Zentner C: Severe neonatal citrullinemia. Arch Dis Child 49:579, 1974.

34. Vidailhet \1, Levin B, Dautrevous M, et a!: Citrulli-nemie. Arch Fr Pediatr 28:521, 19?1.

35. McMurray WC, Rathbun JC, Mohyuddin F, Koegler SJ: Citrullinemia. Pediatrics 32:347, 1963.

36. Morrow C, Barness LA, Efron ML: Citrullinemia with defective urea production. Pediatrics 40:565,

1967.

37. Miyazaki M, Fukuda 5, Aki M, et a!: A case of hepatic encephalomyelopathy associated with citrulline-mia. Brain Nerve 23:19, 1971.

38. Scriver CR, Davies E: Endogenous renal clearance rates

of free amino acids in pre-pubertal children. Pedia-tries 36:592, 1965.

ACKNOWLEDGMENTS

Dr. Nancy Kennaway, University of Oregon Medical

School, performed the assay for argininosuccinic acid

(8)

1976;57;935

Pediatrics

Donald T. Whelan, Terry Brusso and Marilyn Spate