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Cerebrospinal fluid values for monoamine metabolites, γ-aminobutyric acid, and other amino compounds in Rett syndrome
Cerebrospinal fluid values for monoamine metabolites, ,-aminobutyric acid, and other amino compounds in Rett syndrome Thomas L. Perry, MD, Henry G. Dunn, MB, FRCP(C), Helena H. Ho, MD, FRCP(C), a n d John U. Crichton, MB, FRCP(E) From the Department of Pharmacology and Therapeutics and the Department of Paediatrics, University of British Columbia, Vancouver, British Columbia, Canada
We measured concentrations of 3-methoxy.4-hydroxy.phenylglycol, 3,4.dihydroxyphenylacetlc acid, homovanillic acid, and 5-hydroxyindoleacetic a c l d ~ t h e metabolltes of noradrenallne, dopamlne, and serotonln used as central neurotransmittersmln the cerebrosplnal fluid (CSF) specimens of five girls with Rett syndrome. These patients met the clinical criteria for both Inclusion and exclusion of the diagnosis of Rett syndrome. In contrast to previous reports, cerebral monoamlne metabolltes were present In normal concentrations in CSF. In addltlon, concentrations of ~-amlnobutyric acid and of a largenumber of other amino acids and related compounds were normal in the CSF of patients with the syndrome. We doubt that an underlying biochemical cause for this disorder has yet been dlscovered. (J PEDIATR1988;112:234-8)
Rett syndrome is a severe, progressive brain disorder occurring only in girls, and it is possibly as common as phenylketonuria? "s Normal neurologic development is present during the first 6 to 18 months of life, followed by failure to acquire new skills at the appropriate rate and a loss of skills already acquired, and then rapid deterioration of behavior and mental status. Patients lose purposeful use of the hands, develop some characteristics of autism, stereotyped hand movements, and truncal apraxia-ataxia, and become severely demented. Head circumference, whieh is normal in early infancy, becomes re!atively small, presumably because of impaired brain growth. Episodic hyperventilation is common, and epilepsy eventually develops in 75% to 80% of patients. It has been suggested 4 that Rett syndrome may be caused by a dominant mutation Supported by a grant to Dr. Perry from the Medical Research Council of Canada. Submitted for publication July 27, 1987; accepted Sept. 11, 1987. Reprint requests: Thomas L. Perry, MD, Department of Pharmacology and Therapeutics, Vancouver, British Columbia, V6T 1W5, Canada
234
occurring on one X chromosome during gametogenesis, with the hemizygous state in males being lethal for the fetus, so that the syndrome is recognized only in girls. Hagberg et al. 4 found no consistent laboratory abnormalities that might explain the failure of brain growth and development in 35 patients with Rett syndrome. Hyperammonemia, originally thought to be a biochemical feature of MHPG HVA CSF 5-HIAA GABA
3-Methoxy-4-hydroxyphenylglycol Homovanillicacid Cerebrospinalfluid 5-Hydroxyindoleacetic acid -g-Aminobutyrieacid
the syndrome, 2,3 was present in only four of 23 patients. However, Zoghbi et al. 6 later reported a significant reduction in the mean concentrations of 3-methoxy-4-hydroxy phenylglycol and homovanillic acid, which are cerebral metabolites of noradrenaline and dopamine, respectively, in the cerebrospinal fluid of six patients with Rett syndrome. More recently, these authors described reductions in levels of MHPG, HVA, and 5-hydroxyindoleacetic acid
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CFS values for amino compounds in Rett syndrome
235
T a b l e I. Clinical characteristics of patients with Rett syndrome
Age at end of normal Age development Patient (yr) (too) 1 2 3 4 5
16.0 10.5 7.0 6.0 4.0
Age at onset Age at loss of definite of purposeful regression hand use (mo) (too)
15 3 9 6 10
24 18 17 8 16
<48 24 34 33 20
Onset of truncal Latest head ataxia clrcumference (yr) (perCentile) <4.0 5.5 5.0 <4.0 <4.0
EEG abnormalities appeared (yr)
Onset of first eplleptlc seizures (yr)
2.5 5.5 1.5 3.0 2.0
2 2 2 10 2
6.0 5.0 <2.5
EEG, electroencephalogram.
T a b l e II. Cerebrospinal fluid concentrations of monoamine metabolites
Compound
Control adults (n = 94, 16-86 yr)
MHPG DOPAC HVA 5-HIAA
7.8 • 3.0 1.6 • 0.9 35.3 • 17.3 20.0 __. 10.1
,,
t
Control children (n = 17, 2-12 yr) ,
Patient I (45 yr)
Patient 2 (10 yr)
7.2 0.5 67.3 22.4
8.3 1.1 74.4 26.4
,
.
9.4 _+ 2.0 2.5 • 2.1 95.7 • 38.0 36.2 _+ 16.9
.
Patient 3 (7 yr) .
.
.
.
.
.
.
8.3 1.3 68.2 18.1
.
Patient 4 (5 yr) , , ,
,
.
9.2 6.8 114.3 31.4
.
.
Patient 5 (2.5 yr) .
.
,
,
14.2 2.1 114.3 28.7
Valuesof metabolitesare expressedin nanogramsper milliliter.(To convertto nanomolesper liter, multiplyby 5.43for MHPG, 5.95for DOPAC,5.49for HVA, ~nd 5.23 for 5-HIAA). Data for controladultsand childrenare means+ SD. ~ges of patientswhenCSF was examinedare indicatedin parentheses. DOPAC,3,4-dihydroxyphenylaeeticacid.
(a serotonin metabolite) in the CSF. They also found elevations in CSF of biopterin, a precursor of tetrahydrobiopterin, which is the active cofactor for the hydroxylation of tyrosine and tryptophan and is thus essential for the synthesis in brain tissue of the three monoamines.7 Other investigators, however, found that concentrations of tetrahydrobiopterin, together with activities of dihydropteridine reductase, the enzyme that synthesizes this cofactor, are normal in patients with Rett syndrome.8 Finally, postmortem studies of brain tissue from a patient with Rett syndrome showed, in most regions, a severe reduction of noradrenaline, dopamine, and serotonin, in comparison with levels present in the brain of an agematched control subjectg; ratios of amines to metabolites indicated increased turnover of dopamine and serotonin. These last investigators9 suggested that a defect in maturation processes of central monoaminergic systems could be the underlying cause of Rett syndrome? We describe here the neurochemical findings in the CSF of five girls who appear to have Rett syndrome, in comparison with those in age-matched control values. METHODS A presumptive diagnosis of Rett syndrome was made in each of five girls whose symptoms met most of the criteria
for inclusion described by Hagberg et al. 4,5 (Table I). None of our patients had prenatally or perinatally acquired brain impairment or congenital microcephaly, and none had visceromegaly, retinopathy, or optic atrophy. 5 All five patients have siblings, none of whom have had any neuropsychiatric disorder. All five patients have normal hearing. The CSF specimens were obtained from the patients with Rett syndrome and from child control subjects, ~vith informed consent from parents, and from adult control subjects with informed consent from either patients or their next of kin. None of the children and few of the adults whose CSF specimens were used to establish control values for GABA, other amino acids, and monoamine metabolites were "normal" subjects. Most had some form of neurologic or psychiatric illness. However, in calculating the control data, we excluded all subjects with diseases known to involve abnormalities of brain amino acids or monoamines, as well as all CSF specimens obtained from patientstaking drugs likely to alter concentrations of any of these compounds. The first 4.5 ml of CSF withdrawn at lumbar puncture was used for analysis of amino acids and 3'aminobutyric acid, and the next aliquot of 5 ml of CSF was used for measuring monoamine metabolites, These same fractions of CSF were used for patients with Rett syn-
236
Perry et al.
The Journal of Pediatrics February 1988
T a b l e III. Concentrations of amino acids and related compounds in C S F
Compound*
Taurine Phosphoethanolamine Threonine Serine Asparagine Glutamic Acid Glutamine Glycine Alanine Citrulline a-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ethanolamine Ornithine Lysine Histidine Homocarnosine Arginine 3,-Aminobutyric acid]
Control children (n = 54, 2-12 yr)
5.4 _+ 2.1 4.1 + 2.3 22.8 _+ 7.7 30.9 _+ 5.6 4.7 + 2.5 0.3 _+ 0.3 497.3 +_ 85.8 4.3 +_ 3.5 23.8 _+ 8.1 1.1 + 0.8 1.8 _+ 1.1 14.9 _+ 5.4 1.9 _+ 0.7 4.0 _+ 1.7 10.8 _+ 3.1 8.5 + 2.8 7.3 + 2.1 16.8 _+ 9.1 3.6 _+ 1.7 20.4 _+ 4.9 12.7 _+ 3.1 4.9 _+ 2.8 18.3 _+ 3.6 (n = 21, 2-12 yr) 101 _+ 51
Patient I
Patient 2
Patient 3
Patient 4
Patient 5
5.3 3.0 35.2 24.8 5.1 0.1 487.2 2.8 16.9 0.8 0.8 13.0 2.5 2.7 9.0 8.8 6.6 4.9 1.8 24.8 14.6 2.3 18.1
4.6 1.0 20.2 25.3 0.9 0.1 422.6 1.3 32.6 0.1 0.4 19.1 1.8 5.2 14.0 13.6 5.8 12.5 2.6 22.4 5.9 trace 13.1
6.6 1.8 9.8 23.6 1.5 0.3 424.2 2.2 16.1 0.2 0.5 12.5 0.5 2.5 8.9 6.8 4.4 5.8 2.0 11.5 8.0 1.4 11.5
3.9 2.0 19.4 36.6 1.5 trace 522.6 4.3 24.8 0.2 4.5 9.7 1.1 1.7 6.2 5.8 5.8 6.1 1.3 9.8 10.4 3.4 13.0
4.5 1.9 18.7 29.0 2.1 trace 426.5 1.9 18.4 0.8 0.8 14.0 1.3 4.0 12.5 10.9 8.5 7.6 2.6 27.4 9.1 3.4 15.7
136
132
29
37
17
*Values are expressed in mieromolesper liter, with means _+SD shown for control subjects. Values are not listed for eight compounds usually detected in CSF in concentrations of 0.2 gmol/L or less: aspartic acid, proline, cystine, tryptophan, oN-methyllysine, 1-methylhistidine,3-methylhistidine, and 3,-aminobutyryllysine. None of the patients with Rett syndrome exhibited increased concentrations of any of these eight compounds. ~'Values are expressed in nanomoles per liter.
d r o m e a n d for all control subjects to avoid errors t h a t m i g h t be introduced by the cephalocaudal gradient t h a t exists for m a n y constituents in h u m a n CSF. M o n o a m i n e metabolites in C S F were determined by h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y with electrochemical detection, with a slight modification of a previously described method. 1~ T h e G A B A concentrations in C S F were measured as described by Perry et al., ~ who used postcolumn derivatization with o - p h t h a l a l d e h y d e and fluorescence detection of the resulting adducts. C S F specimens were deproteinized with sulfosalicylic acid instantly, as they dripped from the l u m b a r p u n c t u r e needle, and were frozen immediately at - 7 0 ~ C to avoid artifactual release of free G A B A from bound forms of G A B A , particularly homocarnosine, which are normally present in CSF. All other amino acids and related amino compounds present in C S F specimens were q u a n t i t a t e d on an a u t o m a t ic a m i n o acid analyzer (Technicon I n s t r u m e n t Corp., Tarrytown, N . Y . ) with the use of a single long column with a lithium citrate elution buffer system. N i n h y d r i n was used for detection? 2, ~3
RESULTS T h e C S F concentrations of M H P G (derived from noradrenaline in brain tissue), 3,4-dihydroxyphenylacetic acid and H V A (metabolites of cerebral dopamine), and 5H I A A (derived from serotonin in brain) did not differ significantly in patients with R e t t syndrome from those in control patients (Table II). Concentrations of H V A and 5 - H I A A are considerably higher in the C S F of infants and young children t h a n in the C S F of older children and adultst*; thus the metabolite values for patients 2 to 5 can properly be compared with the means for the control children, and those for patient 1 m a y be compared with either the child or adult control subjects. N o consistent abnormalities of the concentrations of 23 amino acids and related amines a n d peptides t h a t can be accurately q u a n t i t a t e d on an a u t o m a t i c amino acid analyzer were found in the C S F of the patients with R e t t syndrome (Table III), nor were there excesses in their C S F of eight additional compounds (see footnote to Table III) present in concentrations too low for accurate measurement with a conventional amino acid analyzer technique. T h e r e was no evidence of any a b n o r m a l i t y in the patients'
Volume 112 Number 2
CSF specimens involving the neurotransmitters glycine and glutamic acid, nor the possible neurotransmitters aspartic acid and taurine. All GABA concentrations in the CSF of the patients with Rett syndrome were well within 2 SD of the mean for the control children (Table III). However, all of the control subjects had some form of brain disease. CSF GABA values are not available for well infants and children, and they might differ appreciably from the control mean given in Table IIl. (Our mean CSF GABA concentration for 38 adults without any neurologic or psychiatric disorder is 84 + 36 nmol/L.) DISCUSSION The normal concentrations of metabolites of noradrenaline, dopamine, and serotonin in the CSF of our five patients with clinical characteristics consistent with a diagnosis of Rett syndrome do not support the concept that a defect in maturation processes of monoamine-utilizing neurons in the brain underlies this disorder. Harris et al) 5 also described a 25-year-old woman with Rett syndrome whose CSF concentrations of these brain monoamine metabolites were normal. Our findings in this small group of patients suggest normal formation, release, and turnover of these three biogenic amines. Further careful quantitation of monoamines and their metabolites, at autopsy, in brains of patients who have died from Rett syndrome, like the single case Studied by Briicke et al.,9 should help answer this question. We cannot explain the difference between our finding of normal levels of monoamine metabolites in the CSF of patients with Rett syndrome and the significant reductions in these levels, especially of MHPG and HVA, reported by Zoghbi et al. 6,7 These investigators and we have both used the same clinical criteria for inclusion and exclusion of the diagnosis of Rett syndrome.4'5 Zoghbi et al. 6'7 used gas chromatography-mass spectrometry to measure monoamine metabolites in CSF, whereas we employed highperformance liquid chromatography with electrochemical detection. Our monoamine metabolite values for the CSF of control children agree reasonably well with those reported by other investigators)4'16'17 We doubt that our use of the aliquot of CSF from 4.5 to 9.5 ml obtained at lumbar puncture, rather than the first 2 ml used by Zoghbi et al.,6,7 could account for the discrepancy in results. Not only is there a lack of agreement between ourselves and Zoghbi et al:.7 regarding a reduction of monoamine metabolites present in the CSF of patients with Rett syndrome, but these investigators' results, which suggest a deficiency of tetrahydrobiopterin in Rett syndrome,7 also conflict with the findings of Sahota et al.,8 indicating normal synthesis of tetrahydrobiopterin. Were there a deficiency in the synthesis of this cofactor in Rett syn-
CFS values for amino compounds in Rett syndrome
237
drome, one would anticipate some failure in the hydroxylation of phenylalanine to tyrosine, such as that occurring in patients with hyperphenylalaninemia with diminished dihydropteridine reductase activity. None of our patients with Rett syndrome had an elevated concentration of phenylalanine in CSF. No elevation of the glutamine level was evident in any of the patients' CSF specimens. This finding supports the conclusion of Hagberg et al. 4 that hyperammonemia, originally reported in patients with Rett syndrome,2'3is not a biochemical feature of this disorder. The normal concentrations of a wide variety of other amino acids in CSF exclude many known amino acid metabolic disorders. The probably normal concentrations of GABA in our patients' CSF specimens do not suggest a cerebral abnormality of this important inhibitory neurotransmitter, although normal CSF GABA levels do not rule out an important deficiency of GABA in certain brain regions, as is the case in Huntington chorea." The problem of exploring the possible biochemical basis of Rett syndrome is made difficult by the lack of a specific diagnostic marker for the disease, so that different investigators may inadvertently be studying different disorders. At this point, however, we doubt that any biochemical abnormalities have been clearly established as characteristic of Rett syndrome. We thank Shirley Hansen, Karen Jones, and Catriona Wall for their excellent technical assistance. REFERENCES
1. Hagberg B. Rett-syndrome: prevalence and impact on progressive severe mental retardation in girls. Acta Pediatr Scand 1985;74:405-8. 2. Rett A. Uber ein eigenartiges hirnatrophisches Syndrom bei Hyperammonamie im Kindesalter. Wien Med Wochenschr 1966;116:723-6. 3. Rett A. Cerebral atrophy associated with hyperammonemia. In: Vinken PJ, Bruyn GW, eds. Handbook of clinical neurology; vo129. Amsterdam: North Holland (Elsevier), 1977:30529. 4. Hagberg B, Aicardi J, Dias K, Ramos O. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett syndrome--report of 35 cases. Ann Neurol 1983;14:471-9. 5. Hagberg B, Gouti6res F, Hanefeld F, Rett A, Wilson J. Rett syndrome: criteria for inclusion and exclusion. Brain Dev 1985;7:372-3. 6. Zoghbi HY, Percy AK, Glaze DG, Butler IJ, Riccardi VM. Reduction of biogenic amine levels in the Rett syndrome. N Engl J Med 1985;313:921-4. 7. Zoghbi HY, Milstein S, Butler IJ, Percy AK. Cerebrospinal fluid biogenic amines and biopterin in Rett syndrome. Ann Neurol 1986;20:433-4. 8. Sahota A, Leeming R, Blair J, Hagberg B. Tetrahydrobiopterin metabolism in the Rett disease. Brain Dev 1985;7:34950. 9. Briicke T, Sofic E, Killian W, Rett A, Riederer P. Reduced
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10.
11.
12.
13.
Perry et al.
The Journal of Pediatrics February 1988
concentrations and increased metabolism of biogenic amines in a single case of Rett-syndrome: a postmortem brain study. J Neural Transm 1987;68:315-24. Perry TL, Yong VW, Ito M, et al. 1-Methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig. Life Sci 1985; 36:1233-8. Perry TL, Hansen S, Wall RA, Gauthier SG. Human CSF GABA concentrations: reviseddownwardfor controls, but not decreased in Huntington's chorea. J Neurochem 1982;38:76673. Perry TL, Stedman D, Hansen S. A versatile lithium buffer elution system for single column automatic amino acid chromatography. J Chromatogr 1968;38:460-6. Perry TL, Hansen S, Kennedy J. CSF amino acids and plasma-CSF amino acid ratios in adults. J Neurochem 1975;24:587-9.
14. Langlais PJ, Walsh FX, Bird ED, Levy HL. Cerebrospinal fluid neurotransmitter metabolites in neurologically normal infants and children. Pediatrics 1985;75:580-6. 15. Harris JC, Wong DF, Wagner HN Jr, et al. Positron emission tomographic study of Dz dopamine receptor binding and CSF biogenic amine metabolites in Rett syndrome. Am J Med Genet 1986;24(suppl 1):201-10. 16. Butler IJ, Koslow SH, Seifert WE Jr, Caprioli RM, Singer HS. Biogenic amine metabolism in Tourette syndrome. Ann Neurol 1979;6:37-9. 17. Shawitz BA, Cohen DJ, Bowers MB Jr. CSF monoamine metabolites in children with minimal brain dysfunction: evidence for alteration of brain dopamine. J PEDIATR 1977;90:67-7I.
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We measured concentrations of 3-methoxy.4-hydroxy.phenylglycol, 3,4.dihydroxyphenylacetlc acid, homovanillic acid, and 5-hydroxyindoleacetic a c l d ~ t h e metabolltes of noradrenallne, dopamlne, and serotonln used as central neurotransmittersmln the cerebrosplnal fluid (CSF) specimens of five girls with Rett syndrome. These patients met the clinical criteria for both Inclusion and exclusion of the diagnosis of Rett syndrome. In contrast to previous reports, cerebral monoamlne metabolltes were present In normal concentrations in CSF. In addltlon, concentrations of ~-amlnobutyric acid and of a largenumber of other amino acids and related compounds were normal in the CSF of patients with the syndrome. We doubt that an underlying biochemical cause for this disorder has yet been dlscovered. (J PEDIATR1988;112:234-8)
Rett syndrome is a severe, progressive brain disorder occurring only in girls, and it is possibly as common as phenylketonuria? "s Normal neurologic development is present during the first 6 to 18 months of life, followed by failure to acquire new skills at the appropriate rate and a loss of skills already acquired, and then rapid deterioration of behavior and mental status. Patients lose purposeful use of the hands, develop some characteristics of autism, stereotyped hand movements, and truncal apraxia-ataxia, and become severely demented. Head circumference, whieh is normal in early infancy, becomes re!atively small, presumably because of impaired brain growth. Episodic hyperventilation is common, and epilepsy eventually develops in 75% to 80% of patients. It has been suggested 4 that Rett syndrome may be caused by a dominant mutation Supported by a grant to Dr. Perry from the Medical Research Council of Canada. Submitted for publication July 27, 1987; accepted Sept. 11, 1987. Reprint requests: Thomas L. Perry, MD, Department of Pharmacology and Therapeutics, Vancouver, British Columbia, V6T 1W5, Canada
234
occurring on one X chromosome during gametogenesis, with the hemizygous state in males being lethal for the fetus, so that the syndrome is recognized only in girls. Hagberg et al. 4 found no consistent laboratory abnormalities that might explain the failure of brain growth and development in 35 patients with Rett syndrome. Hyperammonemia, originally thought to be a biochemical feature of MHPG HVA CSF 5-HIAA GABA
3-Methoxy-4-hydroxyphenylglycol Homovanillicacid Cerebrospinalfluid 5-Hydroxyindoleacetic acid -g-Aminobutyrieacid
the syndrome, 2,3 was present in only four of 23 patients. However, Zoghbi et al. 6 later reported a significant reduction in the mean concentrations of 3-methoxy-4-hydroxy phenylglycol and homovanillic acid, which are cerebral metabolites of noradrenaline and dopamine, respectively, in the cerebrospinal fluid of six patients with Rett syndrome. More recently, these authors described reductions in levels of MHPG, HVA, and 5-hydroxyindoleacetic acid
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CFS values for amino compounds in Rett syndrome
235
T a b l e I. Clinical characteristics of patients with Rett syndrome
Age at end of normal Age development Patient (yr) (too) 1 2 3 4 5
16.0 10.5 7.0 6.0 4.0
Age at onset Age at loss of definite of purposeful regression hand use (mo) (too)
15 3 9 6 10
24 18 17 8 16
<48 24 34 33 20
Onset of truncal Latest head ataxia clrcumference (yr) (perCentile) <4.0 5.5 5.0 <4.0 <4.0
EEG abnormalities appeared (yr)
Onset of first eplleptlc seizures (yr)
2.5 5.5 1.5 3.0 2.0
2 2 2 10 2
6.0 5.0 <2.5
EEG, electroencephalogram.
T a b l e II. Cerebrospinal fluid concentrations of monoamine metabolites
Compound
Control adults (n = 94, 16-86 yr)
MHPG DOPAC HVA 5-HIAA
7.8 • 3.0 1.6 • 0.9 35.3 • 17.3 20.0 __. 10.1
,,
t
Control children (n = 17, 2-12 yr) ,
Patient I (45 yr)
Patient 2 (10 yr)
7.2 0.5 67.3 22.4
8.3 1.1 74.4 26.4
,
.
9.4 _+ 2.0 2.5 • 2.1 95.7 • 38.0 36.2 _+ 16.9
.
Patient 3 (7 yr) .
.
.
.
.
.
.
8.3 1.3 68.2 18.1
.
Patient 4 (5 yr) , , ,
,
.
9.2 6.8 114.3 31.4
.
.
Patient 5 (2.5 yr) .
.
,
,
14.2 2.1 114.3 28.7
Valuesof metabolitesare expressedin nanogramsper milliliter.(To convertto nanomolesper liter, multiplyby 5.43for MHPG, 5.95for DOPAC,5.49for HVA, ~nd 5.23 for 5-HIAA). Data for controladultsand childrenare means+ SD. ~ges of patientswhenCSF was examinedare indicatedin parentheses. DOPAC,3,4-dihydroxyphenylaeeticacid.
(a serotonin metabolite) in the CSF. They also found elevations in CSF of biopterin, a precursor of tetrahydrobiopterin, which is the active cofactor for the hydroxylation of tyrosine and tryptophan and is thus essential for the synthesis in brain tissue of the three monoamines.7 Other investigators, however, found that concentrations of tetrahydrobiopterin, together with activities of dihydropteridine reductase, the enzyme that synthesizes this cofactor, are normal in patients with Rett syndrome.8 Finally, postmortem studies of brain tissue from a patient with Rett syndrome showed, in most regions, a severe reduction of noradrenaline, dopamine, and serotonin, in comparison with levels present in the brain of an agematched control subjectg; ratios of amines to metabolites indicated increased turnover of dopamine and serotonin. These last investigators9 suggested that a defect in maturation processes of central monoaminergic systems could be the underlying cause of Rett syndrome? We describe here the neurochemical findings in the CSF of five girls who appear to have Rett syndrome, in comparison with those in age-matched control values. METHODS A presumptive diagnosis of Rett syndrome was made in each of five girls whose symptoms met most of the criteria
for inclusion described by Hagberg et al. 4,5 (Table I). None of our patients had prenatally or perinatally acquired brain impairment or congenital microcephaly, and none had visceromegaly, retinopathy, or optic atrophy. 5 All five patients have siblings, none of whom have had any neuropsychiatric disorder. All five patients have normal hearing. The CSF specimens were obtained from the patients with Rett syndrome and from child control subjects, ~vith informed consent from parents, and from adult control subjects with informed consent from either patients or their next of kin. None of the children and few of the adults whose CSF specimens were used to establish control values for GABA, other amino acids, and monoamine metabolites were "normal" subjects. Most had some form of neurologic or psychiatric illness. However, in calculating the control data, we excluded all subjects with diseases known to involve abnormalities of brain amino acids or monoamines, as well as all CSF specimens obtained from patientstaking drugs likely to alter concentrations of any of these compounds. The first 4.5 ml of CSF withdrawn at lumbar puncture was used for analysis of amino acids and 3'aminobutyric acid, and the next aliquot of 5 ml of CSF was used for measuring monoamine metabolites, These same fractions of CSF were used for patients with Rett syn-
236
Perry et al.
The Journal of Pediatrics February 1988
T a b l e III. Concentrations of amino acids and related compounds in C S F
Compound*
Taurine Phosphoethanolamine Threonine Serine Asparagine Glutamic Acid Glutamine Glycine Alanine Citrulline a-Aminobutyric acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ethanolamine Ornithine Lysine Histidine Homocarnosine Arginine 3,-Aminobutyric acid]
Control children (n = 54, 2-12 yr)
5.4 _+ 2.1 4.1 + 2.3 22.8 _+ 7.7 30.9 _+ 5.6 4.7 + 2.5 0.3 _+ 0.3 497.3 +_ 85.8 4.3 +_ 3.5 23.8 _+ 8.1 1.1 + 0.8 1.8 _+ 1.1 14.9 _+ 5.4 1.9 _+ 0.7 4.0 _+ 1.7 10.8 _+ 3.1 8.5 + 2.8 7.3 + 2.1 16.8 _+ 9.1 3.6 _+ 1.7 20.4 _+ 4.9 12.7 _+ 3.1 4.9 _+ 2.8 18.3 _+ 3.6 (n = 21, 2-12 yr) 101 _+ 51
Patient I
Patient 2
Patient 3
Patient 4
Patient 5
5.3 3.0 35.2 24.8 5.1 0.1 487.2 2.8 16.9 0.8 0.8 13.0 2.5 2.7 9.0 8.8 6.6 4.9 1.8 24.8 14.6 2.3 18.1
4.6 1.0 20.2 25.3 0.9 0.1 422.6 1.3 32.6 0.1 0.4 19.1 1.8 5.2 14.0 13.6 5.8 12.5 2.6 22.4 5.9 trace 13.1
6.6 1.8 9.8 23.6 1.5 0.3 424.2 2.2 16.1 0.2 0.5 12.5 0.5 2.5 8.9 6.8 4.4 5.8 2.0 11.5 8.0 1.4 11.5
3.9 2.0 19.4 36.6 1.5 trace 522.6 4.3 24.8 0.2 4.5 9.7 1.1 1.7 6.2 5.8 5.8 6.1 1.3 9.8 10.4 3.4 13.0
4.5 1.9 18.7 29.0 2.1 trace 426.5 1.9 18.4 0.8 0.8 14.0 1.3 4.0 12.5 10.9 8.5 7.6 2.6 27.4 9.1 3.4 15.7
136
132
29
37
17
*Values are expressed in mieromolesper liter, with means _+SD shown for control subjects. Values are not listed for eight compounds usually detected in CSF in concentrations of 0.2 gmol/L or less: aspartic acid, proline, cystine, tryptophan, oN-methyllysine, 1-methylhistidine,3-methylhistidine, and 3,-aminobutyryllysine. None of the patients with Rett syndrome exhibited increased concentrations of any of these eight compounds. ~'Values are expressed in nanomoles per liter.
d r o m e a n d for all control subjects to avoid errors t h a t m i g h t be introduced by the cephalocaudal gradient t h a t exists for m a n y constituents in h u m a n CSF. M o n o a m i n e metabolites in C S F were determined by h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y with electrochemical detection, with a slight modification of a previously described method. 1~ T h e G A B A concentrations in C S F were measured as described by Perry et al., ~ who used postcolumn derivatization with o - p h t h a l a l d e h y d e and fluorescence detection of the resulting adducts. C S F specimens were deproteinized with sulfosalicylic acid instantly, as they dripped from the l u m b a r p u n c t u r e needle, and were frozen immediately at - 7 0 ~ C to avoid artifactual release of free G A B A from bound forms of G A B A , particularly homocarnosine, which are normally present in CSF. All other amino acids and related amino compounds present in C S F specimens were q u a n t i t a t e d on an a u t o m a t ic a m i n o acid analyzer (Technicon I n s t r u m e n t Corp., Tarrytown, N . Y . ) with the use of a single long column with a lithium citrate elution buffer system. N i n h y d r i n was used for detection? 2, ~3
RESULTS T h e C S F concentrations of M H P G (derived from noradrenaline in brain tissue), 3,4-dihydroxyphenylacetic acid and H V A (metabolites of cerebral dopamine), and 5H I A A (derived from serotonin in brain) did not differ significantly in patients with R e t t syndrome from those in control patients (Table II). Concentrations of H V A and 5 - H I A A are considerably higher in the C S F of infants and young children t h a n in the C S F of older children and adultst*; thus the metabolite values for patients 2 to 5 can properly be compared with the means for the control children, and those for patient 1 m a y be compared with either the child or adult control subjects. N o consistent abnormalities of the concentrations of 23 amino acids and related amines a n d peptides t h a t can be accurately q u a n t i t a t e d on an a u t o m a t i c amino acid analyzer were found in the C S F of the patients with R e t t syndrome (Table III), nor were there excesses in their C S F of eight additional compounds (see footnote to Table III) present in concentrations too low for accurate measurement with a conventional amino acid analyzer technique. T h e r e was no evidence of any a b n o r m a l i t y in the patients'
Volume 112 Number 2
CSF specimens involving the neurotransmitters glycine and glutamic acid, nor the possible neurotransmitters aspartic acid and taurine. All GABA concentrations in the CSF of the patients with Rett syndrome were well within 2 SD of the mean for the control children (Table III). However, all of the control subjects had some form of brain disease. CSF GABA values are not available for well infants and children, and they might differ appreciably from the control mean given in Table IIl. (Our mean CSF GABA concentration for 38 adults without any neurologic or psychiatric disorder is 84 + 36 nmol/L.) DISCUSSION The normal concentrations of metabolites of noradrenaline, dopamine, and serotonin in the CSF of our five patients with clinical characteristics consistent with a diagnosis of Rett syndrome do not support the concept that a defect in maturation processes of monoamine-utilizing neurons in the brain underlies this disorder. Harris et al) 5 also described a 25-year-old woman with Rett syndrome whose CSF concentrations of these brain monoamine metabolites were normal. Our findings in this small group of patients suggest normal formation, release, and turnover of these three biogenic amines. Further careful quantitation of monoamines and their metabolites, at autopsy, in brains of patients who have died from Rett syndrome, like the single case Studied by Briicke et al.,9 should help answer this question. We cannot explain the difference between our finding of normal levels of monoamine metabolites in the CSF of patients with Rett syndrome and the significant reductions in these levels, especially of MHPG and HVA, reported by Zoghbi et al. 6,7 These investigators and we have both used the same clinical criteria for inclusion and exclusion of the diagnosis of Rett syndrome.4'5 Zoghbi et al. 6'7 used gas chromatography-mass spectrometry to measure monoamine metabolites in CSF, whereas we employed highperformance liquid chromatography with electrochemical detection. Our monoamine metabolite values for the CSF of control children agree reasonably well with those reported by other investigators)4'16'17 We doubt that our use of the aliquot of CSF from 4.5 to 9.5 ml obtained at lumbar puncture, rather than the first 2 ml used by Zoghbi et al.,6,7 could account for the discrepancy in results. Not only is there a lack of agreement between ourselves and Zoghbi et al:.7 regarding a reduction of monoamine metabolites present in the CSF of patients with Rett syndrome, but these investigators' results, which suggest a deficiency of tetrahydrobiopterin in Rett syndrome,7 also conflict with the findings of Sahota et al.,8 indicating normal synthesis of tetrahydrobiopterin. Were there a deficiency in the synthesis of this cofactor in Rett syn-
CFS values for amino compounds in Rett syndrome
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drome, one would anticipate some failure in the hydroxylation of phenylalanine to tyrosine, such as that occurring in patients with hyperphenylalaninemia with diminished dihydropteridine reductase activity. None of our patients with Rett syndrome had an elevated concentration of phenylalanine in CSF. No elevation of the glutamine level was evident in any of the patients' CSF specimens. This finding supports the conclusion of Hagberg et al. 4 that hyperammonemia, originally reported in patients with Rett syndrome,2'3is not a biochemical feature of this disorder. The normal concentrations of a wide variety of other amino acids in CSF exclude many known amino acid metabolic disorders. The probably normal concentrations of GABA in our patients' CSF specimens do not suggest a cerebral abnormality of this important inhibitory neurotransmitter, although normal CSF GABA levels do not rule out an important deficiency of GABA in certain brain regions, as is the case in Huntington chorea." The problem of exploring the possible biochemical basis of Rett syndrome is made difficult by the lack of a specific diagnostic marker for the disease, so that different investigators may inadvertently be studying different disorders. At this point, however, we doubt that any biochemical abnormalities have been clearly established as characteristic of Rett syndrome. We thank Shirley Hansen, Karen Jones, and Catriona Wall for their excellent technical assistance. REFERENCES
1. Hagberg B. Rett-syndrome: prevalence and impact on progressive severe mental retardation in girls. Acta Pediatr Scand 1985;74:405-8. 2. Rett A. Uber ein eigenartiges hirnatrophisches Syndrom bei Hyperammonamie im Kindesalter. Wien Med Wochenschr 1966;116:723-6. 3. Rett A. Cerebral atrophy associated with hyperammonemia. In: Vinken PJ, Bruyn GW, eds. Handbook of clinical neurology; vo129. Amsterdam: North Holland (Elsevier), 1977:30529. 4. Hagberg B, Aicardi J, Dias K, Ramos O. A progressive syndrome of autism, dementia, ataxia, and loss of purposeful hand use in girls: Rett syndrome--report of 35 cases. Ann Neurol 1983;14:471-9. 5. Hagberg B, Gouti6res F, Hanefeld F, Rett A, Wilson J. Rett syndrome: criteria for inclusion and exclusion. Brain Dev 1985;7:372-3. 6. Zoghbi HY, Percy AK, Glaze DG, Butler IJ, Riccardi VM. Reduction of biogenic amine levels in the Rett syndrome. N Engl J Med 1985;313:921-4. 7. Zoghbi HY, Milstein S, Butler IJ, Percy AK. Cerebrospinal fluid biogenic amines and biopterin in Rett syndrome. Ann Neurol 1986;20:433-4. 8. Sahota A, Leeming R, Blair J, Hagberg B. Tetrahydrobiopterin metabolism in the Rett disease. Brain Dev 1985;7:34950. 9. Briicke T, Sofic E, Killian W, Rett A, Riederer P. Reduced
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The Journal of Pediatrics February 1988
concentrations and increased metabolism of biogenic amines in a single case of Rett-syndrome: a postmortem brain study. J Neural Transm 1987;68:315-24. Perry TL, Yong VW, Ito M, et al. 1-Methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig. Life Sci 1985; 36:1233-8. Perry TL, Hansen S, Wall RA, Gauthier SG. Human CSF GABA concentrations: reviseddownwardfor controls, but not decreased in Huntington's chorea. J Neurochem 1982;38:76673. Perry TL, Stedman D, Hansen S. A versatile lithium buffer elution system for single column automatic amino acid chromatography. J Chromatogr 1968;38:460-6. Perry TL, Hansen S, Kennedy J. CSF amino acids and plasma-CSF amino acid ratios in adults. J Neurochem 1975;24:587-9.
14. Langlais PJ, Walsh FX, Bird ED, Levy HL. Cerebrospinal fluid neurotransmitter metabolites in neurologically normal infants and children. Pediatrics 1985;75:580-6. 15. Harris JC, Wong DF, Wagner HN Jr, et al. Positron emission tomographic study of Dz dopamine receptor binding and CSF biogenic amine metabolites in Rett syndrome. Am J Med Genet 1986;24(suppl 1):201-10. 16. Butler IJ, Koslow SH, Seifert WE Jr, Caprioli RM, Singer HS. Biogenic amine metabolism in Tourette syndrome. Ann Neurol 1979;6:37-9. 17. Shawitz BA, Cohen DJ, Bowers MB Jr. CSF monoamine metabolites in children with minimal brain dysfunction: evidence for alteration of brain dopamine. J PEDIATR 1977;90:67-7I.
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