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Amino acid and enzyme studies of brain and other tissues in an infant with argininosuccinic aciduria
257
Clinica Chimica Acta, 105 (1980) 257-267 0 Else~i~r/North-Hoi~~nd Biomedical Press
CCA 1430
AMINO ACID AND ENZYME STUDIES OF BRAIN AND OTHER TISSUES IN AN INFANT WITH ARGININOSUCCINIC ACIDURIA
THOMAS I.,. PERRY a**, MARY ILK. WIRTZ b, NANCY G. KENNAWAY b, Y. EDWARD HSIA c, FERNANDO C. ATIENZA d and HERBERT S. UEMURA
c
a Department of Pkar~~u~~logy, W~~~vers~ty of British Columbia (Canada), b Division of Medical Genetics, Uniuersity of Oregon Health Sciences Center (U.S.A.], c Departments Medical Genetics, Pediatrics and Pathology, University of Hawaii (Hawaii) nnd ’ Frank Clinic, Honolulu (Hawaii) (Received
December
of
4th, 1979)
Amino acid contents were measured in four regions of autopsied brain from an infant who presented in coma at the age of 7 weeks and died with argininosuccinic aciduria. Argininosuccinic acid lyase activity was greatly reduced in liver, kidney and cultured skin fibroblasts; incorporation of [ %]citrulline into protein by fibroblasts was minimal. Argininosuccinic acid lyase activity in brain was only slightly lower than that in control infant brain. Nevertheless, the brain showed extensive microscopic changes and a marked accumulation of argininosuccinic acid, varying between regions from 1.8 to 4.4 mmol/l. Brain contents of glutamine, glutamic acid, and cu-amino-n-butyric acid were also greatly elevated, with a lesser efevation of citrulline, and a normal arginine content. These studies suggest genetic heterogeneity of tissue enzymes in argininosuccinie aciduria and offer some clues about pathogenesis of the neurological damage often seen in this disorder.
Introduction Argininosuccinic aciduria is probably the most common of the five genetically determined disorders of the urea cycle in man [ 11. It is characterised by absent or decreased activity of argininosuccinic acid lyase (I.,-argininosuccinate arginine-lyase, EC 4.3.2.1, ASA lyase), the fourth sequential enzyme in the urea cycle, with a resulting accumulation of argini~osucci~li~ acid (ASA) in
* Correspondence Columbia,
should
Vancouver,
be V6T
directed lW5,
to
Canada.
T.L.P.
at
Department
of
Pharmacology.
University
of
British
258
tissues and physiological fluids, as well as by mild to marked hyperammonemia [Z]. The clinical picture of ~gininosuccinic aciduria is surprisingly variable. In some affected infants, symptoms including lethargy, poor feeding, tachypnea and seizures may appear within 48 h after birth, and may progress rapidly through coma to death within the first 10 days of life. In other patients, the disease may not become manifest until later in infancy, or until after the second year of life. Some patients have prolonged survival with mental retardation and inte~ittent ataxia. Finally, argininosuccinic aciduria has been completely asymptomatic in at least 11 infants whose disorder was detected by routine urine screening tests [2]. Although the normal progress of these asymptomatic patients may have been the result of appropriate treatment (a low-protein diet, with or without arginine supplements), at least one patient is known who was clinically normal at the age of 3.5 years without any dietary restriction [Z]. The marked variation in the clinical manifestations of ar~ninosuccinic aciduria is compatible with genetic heterogeneity, and studies of the distribution of ASA lyase activity in autopsied tissues strongly suggest that there are several different biochemical forms of the disease [ 31. In this paper, we describe the abnormalities in amino acid content in brain, and the activities of ASA lyase found in liver, kidney, brain, and cultured skin fibroblasts of an infant with argininosuccinic aciduria who was asymptomatic until the age of 7 weeks, when he presented with sudden coma and rapidly died. Such biochemical studies may eventually lead to a better understanding of why this inherited disorder behaves so differently from patient to patient, and how it produces brain damage. Case report The patient was the first child of healthy unrelated Japanese parents. He was born at 38 weeks gestation after an uneventful pregnancy. Birth weight was 3060 g. There was mild jaundice in the newborn period which resolved with phototherapy. He was breast-fed, but was switched to Similac at 5 weeks of age, and then to Isomil, because he was “colicky”. At age 7 weeks, he presented with sudden lethargy, fever, prostration and profuse perspiration, and rapidly became comatose. His weight was 4160 g; length 51 cm; head circumference 37 cm. He was barely responsive to painful stimuli, with occasional purposeless movements of his extremities and increased tendon reflexes. The liver was enlarged. Abnormal laboratory findings included serum bicarbonate 19 mEq/l; chloride 115 mEq/l; calcium 11.6 mg/dl; ammonia 970 ug/dl (normal
259
and he developed hypocalcemia and hyperkalemia. The arterial pH, which had been corrected, rose again to 7.48. Despite vigorous treatment with intravenous fluids, electrolytes, mannitol, increased calories, injections of insulin and anabolic steroids, and use of enteric resins and antibiotics, the liver increased in size, the pupils became fixed and dilated, and the infant died on the fourth day of hospitalization. Two-dimensional thin-layer chromatography of this infant’s urine showed two large ninhydrin-positive spots in the regions of ASA and its anhydrides, as well as an increased concentration of glutamine. Urine, plasma, and CSF were not available for quantitative amino acid analyses. Autopsy specimens of liver, kidney and part of the brain, obtained 150 min after death, were immediately frozen for subsequent studies. A skin biopsy was also obtained. The brain was edematous, with histological evidence of focal necrosis. There was spongy degeneration, particularly in the hypothalamus, caudate nucleus, and adjacent cerebral cortex, but almost sparing the putamen. Generalized Alzheimer type II astrocyte changes were widespread throughout the entire cerebral cortex and subjacent white matter. Methods Collection and processing of tissue samples Autopsy samples from the patient and from control subjects were kept frozen at 60°C to -80°C until analysed. The periods of storage in the freezer were 8 months for the patient, and varied between 1 day and 4 years for the control subjects. Specimens of liver, kidney, and cerebral cortex used for control enzyme assays were obtained within 9 h of death from patients aged 13 days to 10 years. These control subjects had died with diagnoses of tyrosinemia, lactic acidosis, cystinosis, ornithine transcarbamylase deficiency, glycine encephalopathy, methylmalonic acidemia, epilepsy, and brain stem glioma. Autopsy specimens from 11 infants aged 2 days to 7 months, and obtained within 6 h of death, were used for the quantitative amino acid analyses of control brain. These control subjects had died with diagnoses of glycine encephalopathy, methylmalonic acidemia, congenital heart disease, birth trauma, polycystic kidney disease, and undifferentiated mental retardation. Skin fibroblasts from the patient and from seven control subjects were cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum, nonessential amino acids, penicillin (100 units/ml) and gentamycin (50 pg/ml). Cells for enzyme assay were harvested with trypsin/EDTA at 5-10 days past confluency, washed twice with phosphate-buffered saline, and lysed by freezethawing four times in water. Amino acid analyses The ASA contents of the patient’s liver and kidney were determined on a Technicon automatic amino acid analyser, after heating tissue extracts at pH 2.2 at 100°C for 2.5 h [4]. Frozen brain specimens from the patient and from the 11 control infants were homogenized and deproteinized with 0.4 mol/l perchloric acid as described elsewhere [ 5,6]. The deproteinized brain extracts were
260
then subjected to quantitative amino acid analysis on a Technicon automatic amino acid analyser, using the lithium citrate buffer elution system of Perry et al. 171. In these analyses, no effort was made to convert ASA to its anhydrides, since such treatment would have made quantitation of glutamine and of a number of other brain amino compounds impractical. In the system we used, ASA is eluted as four peaks in an otherwise blank region of the chromatogram between phenylalanine and y-aminobutyric acid (GABA). A color factor for ASA was determined by relating the optical density of the internal standard used (norleucine) to the total combined area under the four ASA peaks (ASA and its anhydrides). The same proportionality between the four peaks was observed whether authentic ASA was chromatographed alone, or added to control brain extracts. The incorporation of [ “C]citrulline into fibroblast protein in situ was determined by a modification of the method of Jacoby et al. [S] . After 24-h incubation in arginine-free medium containing 5 bg/ml of ureido-[ 14C]citrulline, the cells were washed thoroughly in cold 0.9% saline, fixed with 5% trichloroacetic acid (TCA), washed again in 5% TCA, followed by ethanol, then air-dried. The fixed cell layer was then dissolved in 2% NaZC03 in 0.1 mol/l NaGH ]9] and aliquots removed for scintillation counting and for protein determination [lo]. Results were expressed as dpm/wg protein. All fibroblast cultures were tested for Mycoplasma contamination [ 111 and were proven negative. Eizzg me assays Tissues were homogenized in distilled water at 4”C, centrifuged at 1000 Xg for 10 min, and the supernatants were used for assays. All assays were linear with time and with protein content. ASA lyase activity was determined as described by Shih et al. [lS] with a final ASA concentration of 5 mmol/l in the fibroblast and liver assays, and 20 mmol/l for brain (cerebral cortex) and kidney. Arginase 19 U (~mol/m~)/ml, was added to the fibroblast and liver assays; 60 U/ml was used for the other tissues. Increased concentrations of arginase gave no further increase in activity. Boiled samples were assayed for blank values to allow for ASA lyase contamination of the added arginase. Urea formation was measured by the method of Ceriotti and Gazzaniga [ 131. ASA synthetase activity was measured by a modification [ 141 of the radiochemical procedure of Schimke 1151, with a final citrultine concentration of 5 mmol/l instead of 10 mmol/l. Results The diagnosis of ~~ninosuccinic aciduria in this patient was suggested by the identification of ASA in the urine by thin-layer chromatography, and was later confirmed by the presence of ASA and its two anhydrides in extracts of liver and kidney. The liver ASA content was 9.5 pmol/g wet weight, while that in kidney was 7.0 pmol/g wet weight. Normally ASA is not detectable in these tissues.
261
Qu~titative amino acid analyses showed ASA present in very high concentrations in each of the four regions of the patient’s brain available for study. Normally no ASA is detectable in either biopsy or autopsy specimens of human brain. Table I shows the contents of ASA and of four other relevant amino acids in the patient’s brain, and in three regions of autopsied brain from a group of 11 control infants. Contents of glutamine and of a-amino-n-butyric acid were greatly elevated in the patient’s brain, while that of glutamic acid was substantially elevated. Citrulline contents are not shown in Table I, because q~~~ltitat.ion of this compound is rarely precise in autopsied (as opposed to biopsied) brain, due to incomplete resolution of citruiline from glutathionecysteine mixed disulfide in the ~hromato~aphic system used /7]. Arginine content was not reduced in the patient’s brain, nor were abnormalities found in the content of 4 known or putative amino acid transmitters other than glutamate: GABA, gfycine, aspartate, and taut-he. Fig. 1 illustrates the im~o~ant differences in amino acid content between a control infant’s brain and the patient’s brain with tracings of the same portion of the chromatograms of 100 mg wet wt of frontal cortex from each. The death-to-freezing intervals, and the duration of storage at -80°C before amino acid analysis, were almost the same for these two brains. Besides the large amount of ASA and the great increase in glutamine content in the patient’s brain, lesser increases in the contents of glutamate, a-amino-?z-butyric acid, and citrulline are atso evident. ASA lyase activity in our patient’s fibroblasts was approxin~a~ly 15% of normal, whereas ASA synthetase was 55% of normal (Table II). In vitro incorporation of [14C]citrulline into protein, which requires intact 4SA synthetase
TABLE
I
SELECTED CASES
AMINO
OF
ACIDS
IN
AUTOPSIED
ARGININOSUCCINIC
BRAIN
OF
THE
--.._--.-
-..-_ Patients
PATIENT
AND
OF
OTHER
Brain
Amino
..-
region -.-
acid
infants
Control
Present
*
case
et al. 1181
Solitaire et al. [ 23 Farriaux Rassin
1
et al. I201 et al.
(18.week * Values succinic
frontal
p&amen-&bus
substantia
cortex
pallidus
n&a
thalamus
ASA
0
Gin
3.90
i 2.69
3.88
i 3.22
7.51
i 1.61
GIU
4.39
* 3.31
4.14
c 2.05
4.11
t 0.98
Aba
0.04
i 0.02
0.05
z+ 0.02
0.04
+ 0.01
Arg
0.15
+ 0.12
0.18
i 0.09
0.49
f 0.35
ASA
4.37
3.07
18.34
22.60
Gh
Carton
REPORTED
ACIDURIA
0
0
1.79
2.95
27.55
21.27 10.72
Gh
8.19
10.33
6.92
Aba
0.64
0.54
0.46
Arg
0.34
AS.4
5.20
(grey
ASA
1.36
(frontal
ASA
1.66
(cortexf
ASA
0.50
(region
0.30
0.34
matter)
3.60
(white
0.45 matter)
cortex)
1241 fetus)
in ~.rmol/g acid:
Gin.
wet
wt.
glutamine:
with
mean
Gfu,
f
S.D.
&utamic
for acid:
unspecified) 11
control
Aba,
infants.
Abbre-viations
cu-amino-n-butyrie
acid:
Arg,
used:
ASA,
arginine.
arginino-
Fig. 1. Tracing of a portion of amino acid analyser chromatograms of 100 mg wet wt of autopsied frontal cortex from a control infant (above) and from the patient with argininosuccinic aciduria (below). Standard abbreviations for amino acids are used, as well as the following: Cit. citrulline; GS-SCY. mixed disulfide of glutathione and cysteine; Aba, a-amino-n-butyric acid; Cysta, cystathionine: Nle, norleucine (internal standard); ASA. argitiosuccinic acid; GABA. y-aminobutyric acid.
263
TABLE UREA IN
II CYCLE
SITU
ENZYME
ACTIVITY
IN CULTURED
SKIN
ASA
synthetase
jmml/mg Patient
AND
ASA
0.045
[ 14C1citrulline
lyase
0.002 f 0.015
0.102
zk 0.024
1410
PROTEIN
+ S.D..
with
range
incorporation
protein
f 479
(551-1940)
fl=6
are mean
INTO
57
(0.078+.155)
n=7 values
[ 14ClCITRULLINE
dpmlpg
(0.031&-0.071)
Control
OF
protein/h
0.025
Controls
INCORPORATION
FIBROBLASTS
n=7
shown
in parentheses.
and ASA lyase, was also severely deficient (Table II). The activities of ASA lyase in our patient’s liver and kidney were both markedly reduced (
TABLE
III
ACTIVITY
OF
UREA
CYCLE ASA
ENZYMES
IN
lyase
pnollmg
TISSUES ASA
synthetase
proteinik
Liver patient
*
patient
**
control
*
undetectable
3.52
0.0008 1.54
+ 0.47
0.97
(1.00-1.82)
f 0.27
(0.57-1.13)
n=3
n=4
Kidney patient
**
0.02
control
**
0.47
* **
0.56
+ 0.17
***
0.61
(0.29-0.63)
k 0.29
(0.30-0.87)
n=3
n=3
Brain patient
**
0.020
control
* *
0.041
0.011 + 0.014
0.0083
(0.024-0.062)
n=7
n=7 * ASA ** ***
ASA
concentration
= 5 mmol/l.
concentration
= 20
Patient’s Control
kidney values
mmol/l.
was inadvertently are mean
+ 0.0063
(0.00244.0210)
f S.D.
thawed with
range
for an unknown shown
period
in parentheses.
(<12
h) during
shipment.
264
‘h
007-
F t$
006.
s ,” 0
005
P D
0.04
q
.._L___---I..
0.2
0
04
06
0.8
--
IO
I2
14
u
I6
IE
Amount of Sampie tmg of Pioteini Fig.
2.
4S.4
fyase
activity
in brain
of
the
patient
and
twu
controls
as a functmn
of
the
amount
of san~le
used.
control brain (Fig. 2). ASA lyase activity in control brain showed no correlation with length of time in storage, the lowest and highest activities (0.024 and 0.062 ismoI/mg protein/h) being obtained on samples which had both been stored at -60°C for 4 years. The brain stored for the shortest duration, 4 months, had an activity of 0.053 pmol/mg protein/h. ASA lyase activity in the one biopsy sample was 0.031 pmol/mg protein/h. Discussion ASA lyase activities found in this patient demonstrate tissue enzyme diversupporting the existence of ASA lyase sity in argininosuccinic aciduria, isozymes in man. The first evidence for such isozymes was the finding by Takahara and Natelson [ 161 of different kinetic properties of human red cell and serum ASA lyases, the latter presumably being derived from liver. Colombo and Bnumgartner Cl71 also demonstrated different kinetic properties of this enzyme from control human liver and kidney, and reported normal ASA lyase activity in kidney from a patient with neonatal argininosuccinic aciduria and presumed deficiency of the liver enzyme. Additionally, Click et al. [3] found normal ASA lyase activity in kidney and brain, but deficient activity in the liver of their patient with neonatal disease. In contrast, several authors have reported deficient activity of this enzyme in all tissues examined from their patients (Table IV). Finally, Ratner’s characterization of beef liver, kidney and brain ASA lyases suggests that these tissue enzymes are very similar and probably identical 121,221.
26.5
TABLE ASA
IV LYASE
NORMAL
ACTIVITY
IN
PATIENTS
and
Carton Van
Farriaux Glick Present
Baumgartner
Heiden
ACIDURIA
Liver
Brain
100
1% OF
REPORTED
et al. [ 19 I
2.1
0 17
et al. 131
135
case
Fibroblast
-
%‘O.fi
et al. 1201
-... -.
[171
et al. Cl81
der
ARGININOSUCCINIC
Ridney
Investigators Colombo
WITH
ACTIVITY)
4.2
0.5 <4
._
3.8
Cl4 0
195
0
49
14
-not
measured.
We have found deficient liver, kidney and fibroblast ASA lyase activity, but near normal activity in brain from our patient with argininosuccinic aciduria. This pattern of tissue activity is distinct from any previously reported. Glick et al. [3] suggested that the instability of ASA synthetase and ASA lyase during storage at -60°C might account for the deficient activities of both these enzymes found in kidney and brain of some patients. The kidney from our patient was thawed, although still cold after shipment, yet its ASA synthetase activity was not reduced. It is therefore unlikely that its deficient ASA lyase activity could be explained by the thawing. Furthermore, we found no decline in ASA lyase activity when control kidney was kept at either 4” or 22°C for 21 h. The patient’s cerebral cortical enzyme activity might be interpreted either as deficient (49% of the mean control value) or normal (at the lower end of the normal range). In either case, the pattern of tissue enzyme activity in this patient is unique, and is consistent with the hypothesis [3] that either more than one structural gene codes for tissue ASA lyases, or that there is a regulator gene mutation in this disease. It is difficult to explain the very high content of ASA in our patient’s brain, in the face of so slight a reduction in ASA lyase activity. Glick et al. [3] found normal ASA lyase in the brain of their patient, but unfortunately the brain ASA content was not determined. It has previously been assumed that the high concentrations of ASA in CSF in this disorder, usually 2 or 3 times higher than those in plasma, result from accumulation of ASA in brain due to deficient ASA lyase activity there. ASA content has been measured in autopsied brain in four other patients with argininosuccinic aciduria, and in all of them it was found markedly elevated (Table I). Brain ASA lyase activity was markedly reduced in two of these patients (18, 20), but was not reported for the other two. The paradox of a marked accumulation of ASA in brain while brain ASA lyase activity was normal or only modestly reduced, as in our patient, was unexpected. One possible explanation, first suggested by Shih ] 21, is increased synthesis of ASA in brain. Blood citrufline levels are also elevated in argininosuccinic aciduria, which might lead to increased transport of citrulline into brain 1251 with subsequent conversion to ASA. The increased production of ASA in brain might then exceed the capacity of the normal enzyme to cleave it.
Measurement of other amino acids in brain provides some clues as to what might cause neurological damage and coma in argininosuccinic aciduria. The contents of glutamic acid and glutamine, particularly the latter, were greatly elevated in our patient’s brain (Table I). Presumably these are secondary to brain ammonia accumulation, with a-ketoglutarate being converted first to glutamate and then to glutamine during ammonia detoxification [ 1,2]. The marked elevation of a-amino-n-butyric acid probably results from a similar reaction between ammonia and a-ketobutyrate. Citrulline content was somewhat elevated in our patient’s brain, as has been reported by others [ 18,231. This could represent increased transport of citrulline from blood to brain, as previously discussed, or alternatively, it could result from the high levels of ASA in brain, by a reversal of ASA synthetase. Finally, we found a normal arginine content in our patient’s brain, as was the case in three other patients [ 18,20,23]. This is surprising, since arginine is produced from ASA by the lyase enzyme. At least, it suggests that sufficient arginine is either transported into brain, or is synthesized in brain by residual ASA lyase activity. The normal arginine content was probably not the result of protein breakdown in brain, because the contents of many other amino acids which are components of protein were not elevated. The mechanism of the neurotoxicity in argininosuccinic aciduria therefore does not appear to be a deficiency of arginine. It may well also not be due simply to large amounts of ASA in brain. If eventually ASA content in brain is found to be greatly elevated in patients without neurological damage, ASA could be excluded as a toxic agent. Death in our patient was most likely due to overwhelming hyperammonemia, but why this only occurred after 7 weeks of apparently normal development is unexplained. Indeed, it is surprising that severe hyperammonemia occurs in argininosuccinic aciduria, when ASA, the accumulated metabolite of ASA lyase deficiency, seems ideally suited for waste nitrogen excretion. It contains both nitrogen atoms destined for urea synthesis, and has a renal clearance equal to the glomerular filtration rate [ 261. Acknowledgements We are grateful to Mr. C. Burkey for assistance with the enzyme assays, to Mrs. Shirley Hansen and Mrs. Maureen Murphy for help with the amino acid analyses of brain, to Dr. T. Thelen for the thin-layer chromatography, and to Dr. N.R.M. Buist and Dr. R.D. Koler for consultation and for reviewing this manuscript. This research was supported by grants to T.L.P. from the Medical Research Council of Canada, to N.G.K. from the National Institutes of Health (AM 17906) and the National Foundation, March of Dimes (l-621), to M.L.K.W. from the N.L. Tartar Research Fellowship Fund, and to Y.E.H. from the National Foundation, March of Dimes (C-297). References 1
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in the
235-247,
Batshaw,
M.L.
nervous
Plenum
Press,
(1979)
Lancet
system. New
In Handbook York
2, 452-454
of
Neurochemistry.
94.
Clinica Chimica Acta, 105 (1980) 257-267 0 Else~i~r/North-Hoi~~nd Biomedical Press
CCA 1430
AMINO ACID AND ENZYME STUDIES OF BRAIN AND OTHER TISSUES IN AN INFANT WITH ARGININOSUCCINIC ACIDURIA
THOMAS I.,. PERRY a**, MARY ILK. WIRTZ b, NANCY G. KENNAWAY b, Y. EDWARD HSIA c, FERNANDO C. ATIENZA d and HERBERT S. UEMURA
c
a Department of Pkar~~u~~logy, W~~~vers~ty of British Columbia (Canada), b Division of Medical Genetics, Uniuersity of Oregon Health Sciences Center (U.S.A.], c Departments Medical Genetics, Pediatrics and Pathology, University of Hawaii (Hawaii) nnd ’ Frank Clinic, Honolulu (Hawaii) (Received
December
of
4th, 1979)
Amino acid contents were measured in four regions of autopsied brain from an infant who presented in coma at the age of 7 weeks and died with argininosuccinic aciduria. Argininosuccinic acid lyase activity was greatly reduced in liver, kidney and cultured skin fibroblasts; incorporation of [ %]citrulline into protein by fibroblasts was minimal. Argininosuccinic acid lyase activity in brain was only slightly lower than that in control infant brain. Nevertheless, the brain showed extensive microscopic changes and a marked accumulation of argininosuccinic acid, varying between regions from 1.8 to 4.4 mmol/l. Brain contents of glutamine, glutamic acid, and cu-amino-n-butyric acid were also greatly elevated, with a lesser efevation of citrulline, and a normal arginine content. These studies suggest genetic heterogeneity of tissue enzymes in argininosuccinie aciduria and offer some clues about pathogenesis of the neurological damage often seen in this disorder.
Introduction Argininosuccinic aciduria is probably the most common of the five genetically determined disorders of the urea cycle in man [ 11. It is characterised by absent or decreased activity of argininosuccinic acid lyase (I.,-argininosuccinate arginine-lyase, EC 4.3.2.1, ASA lyase), the fourth sequential enzyme in the urea cycle, with a resulting accumulation of argini~osucci~li~ acid (ASA) in
* Correspondence Columbia,
should
Vancouver,
be V6T
directed lW5,
to
Canada.
T.L.P.
at
Department
of
Pharmacology.
University
of
British
258
tissues and physiological fluids, as well as by mild to marked hyperammonemia [Z]. The clinical picture of ~gininosuccinic aciduria is surprisingly variable. In some affected infants, symptoms including lethargy, poor feeding, tachypnea and seizures may appear within 48 h after birth, and may progress rapidly through coma to death within the first 10 days of life. In other patients, the disease may not become manifest until later in infancy, or until after the second year of life. Some patients have prolonged survival with mental retardation and inte~ittent ataxia. Finally, argininosuccinic aciduria has been completely asymptomatic in at least 11 infants whose disorder was detected by routine urine screening tests [2]. Although the normal progress of these asymptomatic patients may have been the result of appropriate treatment (a low-protein diet, with or without arginine supplements), at least one patient is known who was clinically normal at the age of 3.5 years without any dietary restriction [Z]. The marked variation in the clinical manifestations of ar~ninosuccinic aciduria is compatible with genetic heterogeneity, and studies of the distribution of ASA lyase activity in autopsied tissues strongly suggest that there are several different biochemical forms of the disease [ 31. In this paper, we describe the abnormalities in amino acid content in brain, and the activities of ASA lyase found in liver, kidney, brain, and cultured skin fibroblasts of an infant with argininosuccinic aciduria who was asymptomatic until the age of 7 weeks, when he presented with sudden coma and rapidly died. Such biochemical studies may eventually lead to a better understanding of why this inherited disorder behaves so differently from patient to patient, and how it produces brain damage. Case report The patient was the first child of healthy unrelated Japanese parents. He was born at 38 weeks gestation after an uneventful pregnancy. Birth weight was 3060 g. There was mild jaundice in the newborn period which resolved with phototherapy. He was breast-fed, but was switched to Similac at 5 weeks of age, and then to Isomil, because he was “colicky”. At age 7 weeks, he presented with sudden lethargy, fever, prostration and profuse perspiration, and rapidly became comatose. His weight was 4160 g; length 51 cm; head circumference 37 cm. He was barely responsive to painful stimuli, with occasional purposeless movements of his extremities and increased tendon reflexes. The liver was enlarged. Abnormal laboratory findings included serum bicarbonate 19 mEq/l; chloride 115 mEq/l; calcium 11.6 mg/dl; ammonia 970 ug/dl (normal
259
and he developed hypocalcemia and hyperkalemia. The arterial pH, which had been corrected, rose again to 7.48. Despite vigorous treatment with intravenous fluids, electrolytes, mannitol, increased calories, injections of insulin and anabolic steroids, and use of enteric resins and antibiotics, the liver increased in size, the pupils became fixed and dilated, and the infant died on the fourth day of hospitalization. Two-dimensional thin-layer chromatography of this infant’s urine showed two large ninhydrin-positive spots in the regions of ASA and its anhydrides, as well as an increased concentration of glutamine. Urine, plasma, and CSF were not available for quantitative amino acid analyses. Autopsy specimens of liver, kidney and part of the brain, obtained 150 min after death, were immediately frozen for subsequent studies. A skin biopsy was also obtained. The brain was edematous, with histological evidence of focal necrosis. There was spongy degeneration, particularly in the hypothalamus, caudate nucleus, and adjacent cerebral cortex, but almost sparing the putamen. Generalized Alzheimer type II astrocyte changes were widespread throughout the entire cerebral cortex and subjacent white matter. Methods Collection and processing of tissue samples Autopsy samples from the patient and from control subjects were kept frozen at 60°C to -80°C until analysed. The periods of storage in the freezer were 8 months for the patient, and varied between 1 day and 4 years for the control subjects. Specimens of liver, kidney, and cerebral cortex used for control enzyme assays were obtained within 9 h of death from patients aged 13 days to 10 years. These control subjects had died with diagnoses of tyrosinemia, lactic acidosis, cystinosis, ornithine transcarbamylase deficiency, glycine encephalopathy, methylmalonic acidemia, epilepsy, and brain stem glioma. Autopsy specimens from 11 infants aged 2 days to 7 months, and obtained within 6 h of death, were used for the quantitative amino acid analyses of control brain. These control subjects had died with diagnoses of glycine encephalopathy, methylmalonic acidemia, congenital heart disease, birth trauma, polycystic kidney disease, and undifferentiated mental retardation. Skin fibroblasts from the patient and from seven control subjects were cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum, nonessential amino acids, penicillin (100 units/ml) and gentamycin (50 pg/ml). Cells for enzyme assay were harvested with trypsin/EDTA at 5-10 days past confluency, washed twice with phosphate-buffered saline, and lysed by freezethawing four times in water. Amino acid analyses The ASA contents of the patient’s liver and kidney were determined on a Technicon automatic amino acid analyser, after heating tissue extracts at pH 2.2 at 100°C for 2.5 h [4]. Frozen brain specimens from the patient and from the 11 control infants were homogenized and deproteinized with 0.4 mol/l perchloric acid as described elsewhere [ 5,6]. The deproteinized brain extracts were
260
then subjected to quantitative amino acid analysis on a Technicon automatic amino acid analyser, using the lithium citrate buffer elution system of Perry et al. 171. In these analyses, no effort was made to convert ASA to its anhydrides, since such treatment would have made quantitation of glutamine and of a number of other brain amino compounds impractical. In the system we used, ASA is eluted as four peaks in an otherwise blank region of the chromatogram between phenylalanine and y-aminobutyric acid (GABA). A color factor for ASA was determined by relating the optical density of the internal standard used (norleucine) to the total combined area under the four ASA peaks (ASA and its anhydrides). The same proportionality between the four peaks was observed whether authentic ASA was chromatographed alone, or added to control brain extracts. The incorporation of [ “C]citrulline into fibroblast protein in situ was determined by a modification of the method of Jacoby et al. [S] . After 24-h incubation in arginine-free medium containing 5 bg/ml of ureido-[ 14C]citrulline, the cells were washed thoroughly in cold 0.9% saline, fixed with 5% trichloroacetic acid (TCA), washed again in 5% TCA, followed by ethanol, then air-dried. The fixed cell layer was then dissolved in 2% NaZC03 in 0.1 mol/l NaGH ]9] and aliquots removed for scintillation counting and for protein determination [lo]. Results were expressed as dpm/wg protein. All fibroblast cultures were tested for Mycoplasma contamination [ 111 and were proven negative. Eizzg me assays Tissues were homogenized in distilled water at 4”C, centrifuged at 1000 Xg for 10 min, and the supernatants were used for assays. All assays were linear with time and with protein content. ASA lyase activity was determined as described by Shih et al. [lS] with a final ASA concentration of 5 mmol/l in the fibroblast and liver assays, and 20 mmol/l for brain (cerebral cortex) and kidney. Arginase 19 U (~mol/m~)/ml, was added to the fibroblast and liver assays; 60 U/ml was used for the other tissues. Increased concentrations of arginase gave no further increase in activity. Boiled samples were assayed for blank values to allow for ASA lyase contamination of the added arginase. Urea formation was measured by the method of Ceriotti and Gazzaniga [ 131. ASA synthetase activity was measured by a modification [ 141 of the radiochemical procedure of Schimke 1151, with a final citrultine concentration of 5 mmol/l instead of 10 mmol/l. Results The diagnosis of ~~ninosuccinic aciduria in this patient was suggested by the identification of ASA in the urine by thin-layer chromatography, and was later confirmed by the presence of ASA and its two anhydrides in extracts of liver and kidney. The liver ASA content was 9.5 pmol/g wet weight, while that in kidney was 7.0 pmol/g wet weight. Normally ASA is not detectable in these tissues.
261
Qu~titative amino acid analyses showed ASA present in very high concentrations in each of the four regions of the patient’s brain available for study. Normally no ASA is detectable in either biopsy or autopsy specimens of human brain. Table I shows the contents of ASA and of four other relevant amino acids in the patient’s brain, and in three regions of autopsied brain from a group of 11 control infants. Contents of glutamine and of a-amino-n-butyric acid were greatly elevated in the patient’s brain, while that of glutamic acid was substantially elevated. Citrulline contents are not shown in Table I, because q~~~ltitat.ion of this compound is rarely precise in autopsied (as opposed to biopsied) brain, due to incomplete resolution of citruiline from glutathionecysteine mixed disulfide in the ~hromato~aphic system used /7]. Arginine content was not reduced in the patient’s brain, nor were abnormalities found in the content of 4 known or putative amino acid transmitters other than glutamate: GABA, gfycine, aspartate, and taut-he. Fig. 1 illustrates the im~o~ant differences in amino acid content between a control infant’s brain and the patient’s brain with tracings of the same portion of the chromatograms of 100 mg wet wt of frontal cortex from each. The death-to-freezing intervals, and the duration of storage at -80°C before amino acid analysis, were almost the same for these two brains. Besides the large amount of ASA and the great increase in glutamine content in the patient’s brain, lesser increases in the contents of glutamate, a-amino-?z-butyric acid, and citrulline are atso evident. ASA lyase activity in our patient’s fibroblasts was approxin~a~ly 15% of normal, whereas ASA synthetase was 55% of normal (Table II). In vitro incorporation of [14C]citrulline into protein, which requires intact 4SA synthetase
TABLE
I
SELECTED CASES
AMINO
OF
ACIDS
IN
AUTOPSIED
ARGININOSUCCINIC
BRAIN
OF
THE
--.._--.-
-..-_ Patients
PATIENT
AND
OF
OTHER
Brain
Amino
..-
region -.-
acid
infants
Control
Present
*
case
et al. 1181
Solitaire et al. [ 23 Farriaux Rassin
1
et al. I201 et al.
(18.week * Values succinic
frontal
p&amen-&bus
substantia
cortex
pallidus
n&a
thalamus
ASA
0
Gin
3.90
i 2.69
3.88
i 3.22
7.51
i 1.61
GIU
4.39
* 3.31
4.14
c 2.05
4.11
t 0.98
Aba
0.04
i 0.02
0.05
z+ 0.02
0.04
+ 0.01
Arg
0.15
+ 0.12
0.18
i 0.09
0.49
f 0.35
ASA
4.37
3.07
18.34
22.60
Gh
Carton
REPORTED
ACIDURIA
0
0
1.79
2.95
27.55
21.27 10.72
Gh
8.19
10.33
6.92
Aba
0.64
0.54
0.46
Arg
0.34
AS.4
5.20
(grey
ASA
1.36
(frontal
ASA
1.66
(cortexf
ASA
0.50
(region
0.30
0.34
matter)
3.60
(white
0.45 matter)
cortex)
1241 fetus)
in ~.rmol/g acid:
Gin.
wet
wt.
glutamine:
with
mean
Gfu,
f
S.D.
&utamic
for acid:
unspecified) 11
control
Aba,
infants.
Abbre-viations
cu-amino-n-butyrie
acid:
Arg,
used:
ASA,
arginine.
arginino-
Fig. 1. Tracing of a portion of amino acid analyser chromatograms of 100 mg wet wt of autopsied frontal cortex from a control infant (above) and from the patient with argininosuccinic aciduria (below). Standard abbreviations for amino acids are used, as well as the following: Cit. citrulline; GS-SCY. mixed disulfide of glutathione and cysteine; Aba, a-amino-n-butyric acid; Cysta, cystathionine: Nle, norleucine (internal standard); ASA. argitiosuccinic acid; GABA. y-aminobutyric acid.
263
TABLE UREA IN
II CYCLE
SITU
ENZYME
ACTIVITY
IN CULTURED
SKIN
ASA
synthetase
jmml/mg Patient
AND
ASA
0.045
[ 14C1citrulline
lyase
0.002 f 0.015
0.102
zk 0.024
1410
PROTEIN
+ S.D..
with
range
incorporation
protein
f 479
(551-1940)
fl=6
are mean
INTO
57
(0.078+.155)
n=7 values
[ 14ClCITRULLINE
dpmlpg
(0.031&-0.071)
Control
OF
protein/h
0.025
Controls
INCORPORATION
FIBROBLASTS
n=7
shown
in parentheses.
and ASA lyase, was also severely deficient (Table II). The activities of ASA lyase in our patient’s liver and kidney were both markedly reduced (
TABLE
III
ACTIVITY
OF
UREA
CYCLE ASA
ENZYMES
IN
lyase
pnollmg
TISSUES ASA
synthetase
proteinik
Liver patient
*
patient
**
control
*
undetectable
3.52
0.0008 1.54
+ 0.47
0.97
(1.00-1.82)
f 0.27
(0.57-1.13)
n=3
n=4
Kidney patient
**
0.02
control
**
0.47
* **
0.56
+ 0.17
***
0.61
(0.29-0.63)
k 0.29
(0.30-0.87)
n=3
n=3
Brain patient
**
0.020
control
* *
0.041
0.011 + 0.014
0.0083
(0.024-0.062)
n=7
n=7 * ASA ** ***
ASA
concentration
= 5 mmol/l.
concentration
= 20
Patient’s Control
kidney values
mmol/l.
was inadvertently are mean
+ 0.0063
(0.00244.0210)
f S.D.
thawed with
range
for an unknown shown
period
in parentheses.
(<12
h) during
shipment.
264
‘h
007-
F t$
006.
s ,” 0
005
P D
0.04
q
.._L___---I..
0.2
0
04
06
0.8
--
IO
I2
14
u
I6
IE
Amount of Sampie tmg of Pioteini Fig.
2.
4S.4
fyase
activity
in brain
of
the
patient
and
twu
controls
as a functmn
of
the
amount
of san~le
used.
control brain (Fig. 2). ASA lyase activity in control brain showed no correlation with length of time in storage, the lowest and highest activities (0.024 and 0.062 ismoI/mg protein/h) being obtained on samples which had both been stored at -60°C for 4 years. The brain stored for the shortest duration, 4 months, had an activity of 0.053 pmol/mg protein/h. ASA lyase activity in the one biopsy sample was 0.031 pmol/mg protein/h. Discussion ASA lyase activities found in this patient demonstrate tissue enzyme diversupporting the existence of ASA lyase sity in argininosuccinic aciduria, isozymes in man. The first evidence for such isozymes was the finding by Takahara and Natelson [ 161 of different kinetic properties of human red cell and serum ASA lyases, the latter presumably being derived from liver. Colombo and Bnumgartner Cl71 also demonstrated different kinetic properties of this enzyme from control human liver and kidney, and reported normal ASA lyase activity in kidney from a patient with neonatal argininosuccinic aciduria and presumed deficiency of the liver enzyme. Additionally, Click et al. [3] found normal ASA lyase activity in kidney and brain, but deficient activity in the liver of their patient with neonatal disease. In contrast, several authors have reported deficient activity of this enzyme in all tissues examined from their patients (Table IV). Finally, Ratner’s characterization of beef liver, kidney and brain ASA lyases suggests that these tissue enzymes are very similar and probably identical 121,221.
26.5
TABLE ASA
IV LYASE
NORMAL
ACTIVITY
IN
PATIENTS
and
Carton Van
Farriaux Glick Present
Baumgartner
Heiden
ACIDURIA
Liver
Brain
100
1% OF
REPORTED
et al. [ 19 I
2.1
0 17
et al. 131
135
case
Fibroblast
-
%‘O.fi
et al. 1201
-... -.
[171
et al. Cl81
der
ARGININOSUCCINIC
Ridney
Investigators Colombo
WITH
ACTIVITY)
4.2
0.5 <4
._
3.8
Cl4 0
195
0
49
14
-not
measured.
We have found deficient liver, kidney and fibroblast ASA lyase activity, but near normal activity in brain from our patient with argininosuccinic aciduria. This pattern of tissue activity is distinct from any previously reported. Glick et al. [3] suggested that the instability of ASA synthetase and ASA lyase during storage at -60°C might account for the deficient activities of both these enzymes found in kidney and brain of some patients. The kidney from our patient was thawed, although still cold after shipment, yet its ASA synthetase activity was not reduced. It is therefore unlikely that its deficient ASA lyase activity could be explained by the thawing. Furthermore, we found no decline in ASA lyase activity when control kidney was kept at either 4” or 22°C for 21 h. The patient’s cerebral cortical enzyme activity might be interpreted either as deficient (49% of the mean control value) or normal (at the lower end of the normal range). In either case, the pattern of tissue enzyme activity in this patient is unique, and is consistent with the hypothesis [3] that either more than one structural gene codes for tissue ASA lyases, or that there is a regulator gene mutation in this disease. It is difficult to explain the very high content of ASA in our patient’s brain, in the face of so slight a reduction in ASA lyase activity. Glick et al. [3] found normal ASA lyase in the brain of their patient, but unfortunately the brain ASA content was not determined. It has previously been assumed that the high concentrations of ASA in CSF in this disorder, usually 2 or 3 times higher than those in plasma, result from accumulation of ASA in brain due to deficient ASA lyase activity there. ASA content has been measured in autopsied brain in four other patients with argininosuccinic aciduria, and in all of them it was found markedly elevated (Table I). Brain ASA lyase activity was markedly reduced in two of these patients (18, 20), but was not reported for the other two. The paradox of a marked accumulation of ASA in brain while brain ASA lyase activity was normal or only modestly reduced, as in our patient, was unexpected. One possible explanation, first suggested by Shih ] 21, is increased synthesis of ASA in brain. Blood citrufline levels are also elevated in argininosuccinic aciduria, which might lead to increased transport of citrulline into brain 1251 with subsequent conversion to ASA. The increased production of ASA in brain might then exceed the capacity of the normal enzyme to cleave it.
Measurement of other amino acids in brain provides some clues as to what might cause neurological damage and coma in argininosuccinic aciduria. The contents of glutamic acid and glutamine, particularly the latter, were greatly elevated in our patient’s brain (Table I). Presumably these are secondary to brain ammonia accumulation, with a-ketoglutarate being converted first to glutamate and then to glutamine during ammonia detoxification [ 1,2]. The marked elevation of a-amino-n-butyric acid probably results from a similar reaction between ammonia and a-ketobutyrate. Citrulline content was somewhat elevated in our patient’s brain, as has been reported by others [ 18,231. This could represent increased transport of citrulline from blood to brain, as previously discussed, or alternatively, it could result from the high levels of ASA in brain, by a reversal of ASA synthetase. Finally, we found a normal arginine content in our patient’s brain, as was the case in three other patients [ 18,20,23]. This is surprising, since arginine is produced from ASA by the lyase enzyme. At least, it suggests that sufficient arginine is either transported into brain, or is synthesized in brain by residual ASA lyase activity. The normal arginine content was probably not the result of protein breakdown in brain, because the contents of many other amino acids which are components of protein were not elevated. The mechanism of the neurotoxicity in argininosuccinic aciduria therefore does not appear to be a deficiency of arginine. It may well also not be due simply to large amounts of ASA in brain. If eventually ASA content in brain is found to be greatly elevated in patients without neurological damage, ASA could be excluded as a toxic agent. Death in our patient was most likely due to overwhelming hyperammonemia, but why this only occurred after 7 weeks of apparently normal development is unexplained. Indeed, it is surprising that severe hyperammonemia occurs in argininosuccinic aciduria, when ASA, the accumulated metabolite of ASA lyase deficiency, seems ideally suited for waste nitrogen excretion. It contains both nitrogen atoms destined for urea synthesis, and has a renal clearance equal to the glomerular filtration rate [ 261. Acknowledgements We are grateful to Mr. C. Burkey for assistance with the enzyme assays, to Mrs. Shirley Hansen and Mrs. Maureen Murphy for help with the amino acid analyses of brain, to Dr. T. Thelen for the thin-layer chromatography, and to Dr. N.R.M. Buist and Dr. R.D. Koler for consultation and for reviewing this manuscript. This research was supported by grants to T.L.P. from the Medical Research Council of Canada, to N.G.K. from the National Institutes of Health (AM 17906) and the National Foundation, March of Dimes (l-621), to M.L.K.W. from the N.L. Tartar Research Fellowship Fund, and to Y.E.H. from the National Foundation, March of Dimes (C-297). References 1
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