PROTEIN PATTERNS OF THE CEREBROSPINAL FLUID IN CHILDREN WITH CEREBRAL PALSY1 H. Siemes, M. Siegert, D. Rating and F. Hanefeld nKlinische
und Expcrimentelle Plasmaproteinforschung",
Freie Universitat Berlin
Siemes, H., Siegert, M., Rating, D. and Hanefeld, F.: Protein patterns of the cerebrospinal fluid in children with cerebral palsy. Neuropadiatrie 7: 271—282 (1976). Cerebrospinal fluid in 69 children with cerebral palsy (CP) of different etiology was examined by an improved method of agarose-gel electrophoresis. The protein pattern was normal in 12 cases only. In children with CP caused by congenital and postnatal infections raised y-globulin fractions and the appearance of oligoclonal /-globulin bands were the most obvious finding. In patients with CP due to malformations or perinatal brain damage and in children with CP of unknown etiology decreased prealbumin and increased albumin values were combined with hydrocephalus e vacuo in about half of the cases. Less frequent changes of the /^-globulin fractions were found, an increase of the /?l-fraction being more rare than a decrease of the T-globulin. Cerebral palsy hydrocephalus e vacuo albumin /^-globulins
agarose-gel electrophoresis
Introduction Only a few electrophoretic studies of the cerebrospinal fluid (CSF) proteins have been carried out in patients with cerebral palsy (CP) (Bergleiter et al. 1967, Bokonjic and Renders 1964, Habeck 1961, Lowenthal 1964, Schmidt 1972). Most of the investigators agree that protein electrophoresis has little diagnostic value since merely an elevation of Pi-globulinory-globulin in some cases or no changes at all were found. The development of agarose-gel electrophoresis as an improved quantita1
Supported by "Deutsche Forschungsgemeinschaft"
Received: February 2, 1976
y-globulins
prealbumin
tive micro-technique for 1—3 ml CSF has been of great advantage for protein separation in children {Siegert and Siemes, Siemes et al. 1975). The purpose of the present investigations was to study: I. alterations of the CSF protein profile by such a refined electrophoretic technique in children with CP of different etiology; II. interrelations between profile changes and the two frequent CP associated neurological abnormalities hydrocephalus e vacuo and epilepsy, and III. the resulting diagnostic value of CSF electrophoresis.
Accepted: April 2, 1976
Address: H. S., Universitats-Kinderklinik, Heubnerweg 6, D-1000 Berlin 19 Acknowledgement: The authors gratefully acknowledge the expert technical assistence of Frau R. Frommhold.
271
Downloaded by: National University of Singapore. Copyrighted material.
Interdisziplinare Arbeitsgruppe
Vol. 7, No. 3, 1976
Patients and methods A detailed neurological examination had been carried out on 69 children, who showed disorders of the central motor regulation and finally turned out to have CP. The assessment of the clinical data was based upon the clinical records. The classification of the different C P syndromes was: 50 children had hypertonic syndromes, 14 children hypotonic-ataxic syndromes and 5 patients dyskinetic syndromes. Lumbar puncture was performed in these patients as a diagnostic procedure (e. g. exclusion of meningitis in some cases with convulsions, a suspected congenital infection, pneumencephalography in children with a probable malformation of the CNS). Table I shows the distribution of the children according to the most probable etiology. In the group of children with congenital infections the diagnosis was based on clinical aspects and confirmed by serological tests and Table 1 Etiologic classification n Infections of the C N S Congenital infections Postnatal infections (first half-year of life) Malformations Porencephality Hydrancephaly Agenesis of the corpus callosum Perinatal brain damage Unknown etiology
in addition by demonstrating viruses in the urine of the children with cytomegalic inclusion body disease. In the third etiologic group brain damage was caused by severe birth trauma, asphyxia and acidosis. The majority of the children had a severe form of CP. Most of them had mental retardation and 24 children suffered from epilepsy. 43 children, whose head circumference was within normal limits or below the third percentile had hydrocephalus due to cerebral atrophy (hydrocephalus e vacuo), shown either by pneumencephalography in the majority of the children or by echo-encephalography.
After discharge from hospital most of the patients were seen a t regular intervals in the out-patient-department for follow-up. The diagnosis of C P was so confirmed in a few of the very young infants. Total protein of the CSF was determined by a biuret method.' Quantitative agarose-gel electrophoresis was performed as described previously (Siemes et al. 1975). As there is a variation of protein fractions with age it was necessary for evaluating the CSF-electropherograms to refer to the age groups of 54 normal children. The corresponding distribution of the patients with C P was: 14 infants were 4-10 weeks, 43 children 3-23 months and 12 patients 2-6 years of age when lumbar puncture was performed. In order to exclude CSF protein changes due to dysproteinemia serum
Total n = number of cases
I Neuropidiatrie
69
2
Biochemica-Test-Combination, Boehringer Mannheim
GmbH.
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
29
n
V
A
3
TP
2 9
6 2
1
Protein fractions PI A u,
1
(1,
4
(1,'
1
u,"
5
PI
2 5
z Y2
4
Yn
4
?
iVo
v, a
n = Total numbcr of cases; TP = Total Protein; Pi = Prealbumin; A = Albumin, definition of the Globulins according to Lowenthal (1964), yo = oligoclonal ?-Globulin; = number of cases showing increase above or decrease below 2 standard deviations of the mean normal values
Unknown
Perinatal brain damage
Malformation
Postnatal infection
Congenital infection
Etiology of C P
of different etiology
Table 11 Incidence of abnormal total protein concentration and abnormal relative concentrations of protein fractions in children with CP
a
1
n
O,
R
9
0
%
5' 'u
Siemes et al.
Vol. 7, No. 3, 1976
1
NcuropZdiatrie
Downloaded by: National University of Singapore. Copyrighted material.
M K . 9 2 montltr
Fig. 1 CSF-pherogram and its analysis of a 2 months old girl with C P and hydrocephalus due to congenital rubella. PI and PP = Prealbumins, A = Albumin, definition of the Globulins according to Lowenthal (1964). Registration of the Albumin a t 482 nm, the Prealbumins and Globulins a t 610 nm. Pos. = Position of the fractions related to the distance Albumin - application slot (S) = 100
Downloaded by: National University of Singapore. Copyrighted material.
Protein patterns of cerebrospinal fluid
Fig. 2 CSF-pherogram and its analysis of a 3 months old boy with CP and hydrocephalus due to severe asphyxia. For explanation of the symbols see Fig. 1
electrophoresis on cellulose acetate was done simultaneously.
Results I. Alterations of the protein profile according to different etiology The relative concentrations of the fractions were defined abnormal when exceeding two standard deviations of the mean values of 54 normal children reported previously (Siemes et al. 1975). The incidence of abnormal findings in the children of all etiologic groups is summarized in Table II. In all children with C P the relative mobility of the fractions were within normal range. Only in the y3-region additional fractions of oligoclonal yglobulins without a fixed relative mobility appeared in some children. a) Children with CP caused by congenital and postnatal infections
1. Congenital infections Six of the eleven children with congenital infections had cytomegalic inclusion body disease (4 weeks to 18 months of age), three had toxoplasmosis (2'12 to 14 months old) and one child had lues and one rubella, respectively. Fig. 1 shows the CSF-pherogram and its analysis of a child with congenital rubella. N o child of this group had a normal pherogram. There are several remarkable changes: decreased prealbumin in about two thirds of the cases, increased y3-globulin (mainly consisting of I gG) in about 90°/o, an elevated y-trace fraction (y.,)
Vol. 7, No. 3, I976
1
Neuropidiatrie
in ca. three forths and the appearance of oligoclonal yglobulin bands in about one half of the patients. Three of 11 children had an increased az- or ~l-globulin fraction, albumin was lowered in ca. one third of the children. I t should be stressed that only xbout one third of the children had an elevated total protein concentration according to the normal values of different age groups reported by A m m o n and Richterich (1970). 2. Postnatal infections A raised y3-globulin fraction was found in all five children in whom C P was thought to be due to a postnatal infection (8 months to 3 years of age; one child with cytomegalovirus infection, in 4 children a postnatal encephalitis of unknown etiology within the first half-year of life). The 14 months old child with cytomegalic inclusion body disease again showed an oligoclonal protein band in the y-globulin region. A decreased prealbumin was noticed only in one case, whereas two children had an increased prealbumin concentration. A surprising frequent finding was the lowered albumin in three children. b) Children with malformations of CNS
None of the eight children with C P due to malformations ( 3 to 21 months of age) had a normal pherogram. Two of them showed an elevation of total protein and six patients had a lowered prealbumin and an elevated albumin concentration. In 4 children the ysglobulin was increased (see Table 11).
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
Protein patterns of ccrebrospina1 fluid
CP
due to perinatal brain
damage
Only 2 of 16 children with perinatal brain damage showed a normal pherogram. The most obvious abnormaIities were (see Table 11): I . A decrease of prcalbumin or an increase of albumin (about 50 % of the cases) or both (about 40% ). 2. Ambnormal PI-globulin and t-globulin concentrations u p to about 40 % of the children. 3. An clcvated y4-globulin (y-trace) in about 20 5%. Fig. 2 gives an example of a child with CP and hydrocephalus e vacuo due to severe asphyxia. d) Children with CP of unknown etiology
I n this last group of 29 hildren (4 wceks to 6 years of age, see Table 11) 10 had a normal protein profiIe. Again the decrease of prealbumin (ca. 30 %) and increase of albumin (ca. 20 %) were the most obvious changes. Elevation of fll-globulin and lowered r-$0bulin were also found (ca. 15 %). An jnreresting finding seems ro be the
elevation of yJ-globulin (mainly IgG} in 4 children. 11. Relation of protein profile abnormalities to hydrocephalus e vacuo and epilepsy
There was a remarkable association of prealburnin and albumin abnormalities with hydrocephalus e vacuo (see
Table Ill). With exception of the infectious group where in some cases elevated prealbumin andlor decreased albumin values were found hydrocephalus was frequently combined with decreased prealbumin (22 of the 43 cases with hydrocephalus) or increased albumin (20 of the 43 children) or both changes together (15 patients of the 43). Only 3 children without hydrocephalus showed a decreased prealbumin and no child without hydrocephalus had an elevated albumin vaIue. The preal burnin and albumin hanges in the children with hydrocephalus were much more frequent than abnormal total protein concentrations.
Table JII
Incidence of abnormaI total protein concentration and abnormal reIative concencrations of prealbumin and albumin in 43 chiIdren with CP and hydrocephaIus e vacuo
Etiology of
CP
Congenital and posrnatd infections 217 Malformations
318
Pcrinatal brain damage
3!13
Unknown
2/15
317
317 618
2/15
417
317
6/3
6/13
8/13
7/15
6115
1/15
TP = Total Protein, P1 = main Prealbumin fraction, A = Albumin; A, V = increase abovc or decrease below normal range. AP1 + VA, VPI 4-AA = abnormaIities of Prealbumin and Albumin combined, note rhe divergent changes in the infccrious group compared with the others
Downloaded by: National University of Singapore. Copyrighted material.
c) Children with
The protein profiles of children with epilepsy showed no abnormalities that differed horn the ocher patients. Discussion In the present investigation an improved method of a quantitative agarose-geI micro-electropharesjs was applied t o children with CP. Only a few studies on CSF changes in children with CP have been published. The comparison of our results with those of the literature is difficult as the tehniques used for protein electrophoresis vary considerably. Furthermore the age of the patients at the time of lumbar puncture is not documented in most of the publications. Using paper electrophoresis Ha beck (1961) found in 4 out of 9 patients with perinatal brain damage an elevation of (3-globulins and in one case an increased y-globulin. As lowered total protein had been the criteria for sampling the patients the observed abnormalities could be secundary t o this. The CSF of 25 cases with perinatal encephalopathies had been examined by Lowenshal(1964) using agar as well as paper electrophoresis. In patients without epilepsy he observed a certain increase in a-globulins, whereas the normality of fi-globulin vaIues was emphasized. He also noticed that the y-globulin concentrations were normal. In encephalopathies with epilepsy the results were similar, however, the increase of a-globulins was pointed out. Lowenthal came t o the conclusion that the results in perinatal encephaIopathies were normal except in the a-globulins. Bergleiter et al. (1967) reported that
Vol. 7, No. 3, 1916
1
Neuropjdirtric
the paper pherograms in 6 out of 10 paticncs with a pesinatal brain trauma showed increased 8-globulin concentrations. In addition 12 out of 15 patients with cerebral atrophy (demonstrated by pneumencephalography) showed elevated P-gIobulins. Bokon jic and Renders (1964) could not find any abnormalities using agar pherograms in 5 cases with perinatal encephalopathies. Schmidt investigated 90 persons with perinatal trauma by agar-gel electro~horesis. There was no significant change of the CSF protein profile. Not unexpected from the varying results in the literature we found no characteristic pattern for CP. HOWever, one should expect that the CSF protein changes in hildren with CP depend on the cause of thc disorder. By forming groups according to etiology there were several remarkable findings: 1. In children with CP due to congenital and postnatal infections there was a high incidence of elevated yglobulins, particularly 05 the W-globuIin, consisting mainly of Ig G, and the appearance of oligoclonal y-globulin bands.
In infectious diseases of the CNS long standing quantitative and qualitative abnormalities of the y-globulins can not bc attributed ro an increased permeability of the blood-CSF barrier and arc caused by local antibody production within the CNS (Frick and Scheid-Seydel 1958, Cwtler et al. 1970). The discrete antibody bands on the diffuse background of the yJ-gIobulin region are probably caused by restricted
Downloaded by: National University of Singapore. Copyrighted material.
Siemes e t aj.
Protein patterns of cerebrospinal fluid
In our cases this probably characteristic abnormality could be demonstrated up to 18 months of age when cell cound and total protein concentration were normal. 2. In some children with malformations and others with C P of unknown origin the y3-globulin concentration was elevated. This might suggest that an undiagnosed congenital infection was the cause of CP. As the proteins of the y-trace (y,) fraction do not belong to the immunoglobulins the significance of their elevation in some children remains uncertain. 3. Decreased prealbumin andlor increased albumin values were combined with hydrocephalus in about one half of the cases. Compared with ventricular and cisternal fluid spinal subarachnoid fluid of normal individuals shows a lower prealbumin and a higher albu-
min concentration based on regional differences of CSF circulation along the neuraxis. There is a constant protein exchange between the CSF and blood in the absence of local CSF formation in spinal subarachnoid space (Hochwald 1970) which permits albumin to accumulate and s real bum in to decrease. A protein pattern with abnormally decreased prealbumin and increased albumin concentrations can frequently be noticed below an obstructive lesion of the CSF pathway, particularly in spinal subarachnoid space, caused by the persistence of normal protein diffusion while the normal CSF flow is reduced (Matiar-Vahar 1968). According to this observation the similar protein profile in our children with hydrocephalus due to cerebral atrophy may point to a prolonged protein exchange between the CSF and blood due to the enlargement of the CSF pathways with slowed drainage of CSF. The observation of slowed CSF turnover rates demonstrated in adults with normal pressure hydrocephalus would support this hypothesis (Lorenco et a]. 1974). The observations of elevated prealbumin combined with lowered albumin in some children with postencephalitic hydrocephalus points to a different type of CSF hydrodynamics not yet understood. As there is in addition an elevated y3-globulin concentration only in these cases the CSF phorogram indicates the inflammatory origin of hydrocephalus. 4. Apart from the abnormalities of
Downloaded by: National University of Singapore. Copyrighted material.
heterogeneity of the immune response. In the children with congenital infections only a few clones for immunoglobulin production seem to be available as a consequence of immunological immaturity, but the nature of the antigens may be another factor involved (Heremans and Masson 1973). Changes of the electrophoretic protein profile of the CSF in children with congenital infections have not been studied in detail. Only a few cases a t different ages post partum have been reported but in all these children a raised y-globulin concentration was noticed (Arnold and Zetterstrom 1958, Mac/7etanz and H a b e h 1958, Laterre 1965, Siemes 1974).
the prealbumin, albumin and y-globulin concentrations changes of 0,- and 2globulin fractions were noticed in all etiologic groups. The PI-globulin fraction consists mainly of serum transferrin and the 2fraction is made up of a neuraminic acid-deficient transferrin, not found in the serum (Pette and Stupp 1960, Laterre 1965). Frick and Scheid-Seydel (1963) studied intravenously-injected labelled serum transferrin in patients with different diseases of the CNS. Their data indicated that some of the CSF-transferrin was derived immediately from the serum but that an non-labelled pool of C N S was contributing to the CSFtransferrin. When the transferrin concentration was elevated or decreased this seemed to be related to the CNStransferrin pool and the authors postulated an C N S dependant transferrin metabolism. In the 69 children with C P a decrease of the 0,-fraction was found in 8 cases and a lowered 2-fraction in 16 children, 5 of these cases showed both abnormalities combined. As in 9 children there was an association with increased albumin values the lowering of these fractions could be secondary in part of the children. A remarkable change of the P-globulins was a rise of the (31-globulin fraction in 12 of all children with C P (elevation of the z-globulin only in 6 cases). O u r knowledge of the diagnostic significance of elevated P-globulins is rather small, but already in one of the first publications dealing with electro-
Vo1.7, No. 3, 1916
1
Ncuropsdiatrie
phoresis of the CSF one can find the remark that the increase of the PIglobulin points t o a "degenerative" process of brain tissue (Esser and Heinzler 1952). Subsequently this abnormality was found in patients with brain atrophy of different origin, e. g. in cases due to infections and due to brain trauma (Bauer 1953, Bergleiter et al. 1967, Delank 1965, Matiar 1960, Steger 1953). According to these findings one would expect in our study a frequent association of increased 01globulins with hydrocephalus e vacuo, but we found that only 5 out of 12 children with elevated 0,-globulin values had hydrocephalus what meant no coincidence since 43 of the 69 investigated children had hydrocephalus. Machetanz and Habeck (1961) pointed out that an elevation of the PI-globulin concentration was characteristic of the CSF in children suffering from congenital cardiac defects with marked cyanosis, in cases without cyanosis the pherogram was normal.The autors concluded that the PI-globulin increase was due to a "degenerative" process caused by cerebral hypoxia. As no etiologic group was characterized by a decrease or a n increase of one of both S-globulin fractions the meaning of these changes remains unknown. The abnormalities of the y-globulins described in children with CP indicate that electrophoresis of the CSF proteins by an improved technique could be of additional help in diagnosing the origin of this disorder so frequently observed in children.
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
Protein patterns of cerebrospir~alfluid
Zusammenfassung Lumballiquor von 69 Kindern rnit infantiler Zerebralparese verschiedener Genese wurde mit einer verbesserten Methode der Agarosegel-Elektrophorese untersucht. Das Proteinmuster war nur in 12 Fallen normal. Bei Kindern mit einer Zerebralparese infolge einer kongenitalen oder postnatalen Infektion waren erhohte y-Globulinfraktionen und das Auftreten oligoklonaler y-Globulinbanden die bemerkenswertesten Abweichungen. Bei Patienten, bei denen die Zerebralparese durch eine Mii3bildung oder eine perinatale Hirnschadigung und bei Kindern mit einer Zerebralparese unklarer Genese wurden in etwa der Halfte der Falle erniedrigte Praalbumin- und erhohte Albuminwerte in Verbindung mit einem Hydrocephalus e vacuo gefunden. Weniger haufig kamen Veranderungen der 6-Globuline vor, dabei war eine Erhohung der PI-Fraktion seltener als eine Erniedrigung der t-Fraktion. References 1. Ammon, R., Richterich, R.: Die Ermittlung von Normalwerten der Konzentration von Glukose, Protein und Zellen im Liquor des Kindes. Schweiz. med. Wschr. 100: 1317-1320 (1970). 2. Arnold, K. G., Zetterstrom, R.: Proteins in the Cerebrospinal Fluid in the Newborn. Pediatrics 21: 279-387 (1958). 3. Bauer, H . : Uber die Bedeutung der Papier-Elektrophorese des Liquors fiir die klinische Forschung. Dtsch. Z. Nervenheilk. 170: 381-401 (1953).
4. Bergleiter, R., Consbruch, U., Koufen, H . :
Liquor-Beta-Globuline und Hirnatrophie. Arch. Psychiat. Neurol. 210: 140-151 (1967). 5. Bokonjic, R., Renders, J.: Etude electrophorktique de 400 liquides ckphalo-radlidiens pathologiques. Acta Neurol. Belg. 64: 769-776 (1964). 6. Cutler, R. W. P., Watters, G. V., Hammerstadt, J. P.: The origin and turnover rates of cerebrospinal fluid albumin and y-globulin in man. J. neurol. Sci. 10: 259-262 (1970). 7. Delank, H . W.: Das EiweiRbild des Liquor cerebrospinalis und seine klinische Bedeutung. Darmstadt: Steinkopff, 1965. 8. Esser, H., Heinzler, F.: E l e k t r ~ ~ h o r e t i sche EiweiRanalysen im Liquor cerebrospinalis. Dtsch. med. Wschr. 77: 13291330 (1952). 9. Frick, E., Schcid-Seydel, L.: Untersuchungen mit J13'-markiertem Transferrin zur Frage der Abstammung der Liquoreiweif3korper. Klin. Wschr. 41: 589-593 (1963). 10. Frick, E. und Scheid-Seydel, L.: Untersuhungen mit J's1-markicrtem y-Globulin zur Frage der Abstammung der LiquoreiweiRk6rper. Klin. Wschr. 36: 66-69 (1958). 11. Habeck, D.: Zur Verminderung des GesarnteiweiR-Gehaltes im Liquor cerebrospinalis. Arch. Psychiat. Neurol. 202: 354 -370 (1961). 12. Hewmans, J. F. and Masson, P. L.: Specific Analysis of Immunoglobulins. Techniques and Clinical Value. Ciin. Chem. 19: 294-300 (1973). 13. Hochwald, G. M.: Influx of Serum Proteins and their Concentrations in Spinal Fluid along the Neuraxis. J. neurol. Sci. 10: 269-278 (1970). 14. Laterre, E. C.: Lcs protkines du liquide ckphalo-rachidien i l'etat normal et pathologique. Bruxelles: Editions Arscia S. A. 1965. 15. Lorcnco, A. V., Bresnan, M. J., Barlow, Ch. F.: Cerebrospinal Fluid Absorption Deficit in Normal Pressure Hydrocephalus. Arch. Neurol. 30: 387-393 (1974). 16. I.owentha1, A.: Agar Gel Electrophoresis in Neurology. Arnsterdani/New York: Elsevier Publishing Company 1964. 17. Machetanz, E., Habeck, D.: Liquorelektrophoretischc Untersuchungen bei neurologi-
Downloaded by: National University of Singapore. Copyrighted material.
Moreover in part of the children changes of the prealbumin and albumin fractions point t o deviations of the CSF flow caused by hydrocephalus e vacuo.
IS.
19.
20.
21.
22.
schen Erkrankungen im Kindesalter. Zeitschr. Kinderheilk. 81 : 454-471 (1958). Machetanz, E., Habeck, D.: 1st die Hypouie des Zentralnervensystems ein ursachlicher Faktor fiir die p-Globulinvermehrung im Liquor? Arch. Psychiat. Nervenkr. 201: 418-425 (1961). hlatiar, H . : Die Erhohung der p-Globuline irn Liquor. Dtsch. Z. Nervenheilk. 180: 191-215 (1960). Matiar-Vahar, H.: Der lumbale Sperrliquor beim partiellen und kompletten Block. Fortschr. Neurol. Psychiat. 36: 247-257 (1968). Pette, D., Stupp, J.: Die z-Fraktion im Liquor cerebrospinalis. Klin. Wschr. 38: 109-110 (1960). Schmidt, M.: Liquoragargelelektrophorese
Vol. 7, No. 3, 1976
23.
24.
25.
26.
1
Neuropidiatrie
in der neurologisch-psychiatrischen Diagnostik. Jena: VEB Gustav Fischer Verlag 1972. Siegert, M., Siemes, H.: Agarose Gel Electrophoresis of the Proteins of Cerebrospinal Fluid and Analysis of Pherogram Profiles by an Analog Computer. T o be published. Siemes, H., Siegert, M., Lison, H.: Liquorelektrophorese bei connataler Encephalitis. Mschr. Kinderheilk. 122: 440-442 (1974). Siemes, H., Siegert, M. and Rating, D.: Das Liquorproteinprofil normaler Kinder und seine Abhangigkeit vom Lebensalter. Neuropadiatrie 6: 383-397 (1975). Steger, J.: Elektrophoretischc Untersuchungen des Liquors. Dtsch. Z. Nervenheilk. 171: 1-19 (1953).
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
und Expcrimentelle Plasmaproteinforschung",
Freie Universitat Berlin
Siemes, H., Siegert, M., Rating, D. and Hanefeld, F.: Protein patterns of the cerebrospinal fluid in children with cerebral palsy. Neuropadiatrie 7: 271—282 (1976). Cerebrospinal fluid in 69 children with cerebral palsy (CP) of different etiology was examined by an improved method of agarose-gel electrophoresis. The protein pattern was normal in 12 cases only. In children with CP caused by congenital and postnatal infections raised y-globulin fractions and the appearance of oligoclonal /-globulin bands were the most obvious finding. In patients with CP due to malformations or perinatal brain damage and in children with CP of unknown etiology decreased prealbumin and increased albumin values were combined with hydrocephalus e vacuo in about half of the cases. Less frequent changes of the /^-globulin fractions were found, an increase of the /?l-fraction being more rare than a decrease of the T-globulin. Cerebral palsy hydrocephalus e vacuo albumin /^-globulins
agarose-gel electrophoresis
Introduction Only a few electrophoretic studies of the cerebrospinal fluid (CSF) proteins have been carried out in patients with cerebral palsy (CP) (Bergleiter et al. 1967, Bokonjic and Renders 1964, Habeck 1961, Lowenthal 1964, Schmidt 1972). Most of the investigators agree that protein electrophoresis has little diagnostic value since merely an elevation of Pi-globulinory-globulin in some cases or no changes at all were found. The development of agarose-gel electrophoresis as an improved quantita1
Supported by "Deutsche Forschungsgemeinschaft"
Received: February 2, 1976
y-globulins
prealbumin
tive micro-technique for 1—3 ml CSF has been of great advantage for protein separation in children {Siegert and Siemes, Siemes et al. 1975). The purpose of the present investigations was to study: I. alterations of the CSF protein profile by such a refined electrophoretic technique in children with CP of different etiology; II. interrelations between profile changes and the two frequent CP associated neurological abnormalities hydrocephalus e vacuo and epilepsy, and III. the resulting diagnostic value of CSF electrophoresis.
Accepted: April 2, 1976
Address: H. S., Universitats-Kinderklinik, Heubnerweg 6, D-1000 Berlin 19 Acknowledgement: The authors gratefully acknowledge the expert technical assistence of Frau R. Frommhold.
271
Downloaded by: National University of Singapore. Copyrighted material.
Interdisziplinare Arbeitsgruppe
Vol. 7, No. 3, 1976
Patients and methods A detailed neurological examination had been carried out on 69 children, who showed disorders of the central motor regulation and finally turned out to have CP. The assessment of the clinical data was based upon the clinical records. The classification of the different C P syndromes was: 50 children had hypertonic syndromes, 14 children hypotonic-ataxic syndromes and 5 patients dyskinetic syndromes. Lumbar puncture was performed in these patients as a diagnostic procedure (e. g. exclusion of meningitis in some cases with convulsions, a suspected congenital infection, pneumencephalography in children with a probable malformation of the CNS). Table I shows the distribution of the children according to the most probable etiology. In the group of children with congenital infections the diagnosis was based on clinical aspects and confirmed by serological tests and Table 1 Etiologic classification n Infections of the C N S Congenital infections Postnatal infections (first half-year of life) Malformations Porencephality Hydrancephaly Agenesis of the corpus callosum Perinatal brain damage Unknown etiology
in addition by demonstrating viruses in the urine of the children with cytomegalic inclusion body disease. In the third etiologic group brain damage was caused by severe birth trauma, asphyxia and acidosis. The majority of the children had a severe form of CP. Most of them had mental retardation and 24 children suffered from epilepsy. 43 children, whose head circumference was within normal limits or below the third percentile had hydrocephalus due to cerebral atrophy (hydrocephalus e vacuo), shown either by pneumencephalography in the majority of the children or by echo-encephalography.
After discharge from hospital most of the patients were seen a t regular intervals in the out-patient-department for follow-up. The diagnosis of C P was so confirmed in a few of the very young infants. Total protein of the CSF was determined by a biuret method.' Quantitative agarose-gel electrophoresis was performed as described previously (Siemes et al. 1975). As there is a variation of protein fractions with age it was necessary for evaluating the CSF-electropherograms to refer to the age groups of 54 normal children. The corresponding distribution of the patients with C P was: 14 infants were 4-10 weeks, 43 children 3-23 months and 12 patients 2-6 years of age when lumbar puncture was performed. In order to exclude CSF protein changes due to dysproteinemia serum
Total n = number of cases
I Neuropidiatrie
69
2
Biochemica-Test-Combination, Boehringer Mannheim
GmbH.
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
29
n
V
A
3
TP
2 9
6 2
1
Protein fractions PI A u,
1
(1,
4
(1,'
1
u,"
5
PI
2 5
z Y2
4
Yn
4
?
iVo
v, a
n = Total numbcr of cases; TP = Total Protein; Pi = Prealbumin; A = Albumin, definition of the Globulins according to Lowenthal (1964), yo = oligoclonal ?-Globulin; = number of cases showing increase above or decrease below 2 standard deviations of the mean normal values
Unknown
Perinatal brain damage
Malformation
Postnatal infection
Congenital infection
Etiology of C P
of different etiology
Table 11 Incidence of abnormal total protein concentration and abnormal relative concentrations of protein fractions in children with CP
a
1
n
O,
R
9
0
%
5' 'u
Siemes et al.
Vol. 7, No. 3, 1976
1
NcuropZdiatrie
Downloaded by: National University of Singapore. Copyrighted material.
M K . 9 2 montltr
Fig. 1 CSF-pherogram and its analysis of a 2 months old girl with C P and hydrocephalus due to congenital rubella. PI and PP = Prealbumins, A = Albumin, definition of the Globulins according to Lowenthal (1964). Registration of the Albumin a t 482 nm, the Prealbumins and Globulins a t 610 nm. Pos. = Position of the fractions related to the distance Albumin - application slot (S) = 100
Downloaded by: National University of Singapore. Copyrighted material.
Protein patterns of cerebrospinal fluid
Fig. 2 CSF-pherogram and its analysis of a 3 months old boy with CP and hydrocephalus due to severe asphyxia. For explanation of the symbols see Fig. 1
electrophoresis on cellulose acetate was done simultaneously.
Results I. Alterations of the protein profile according to different etiology The relative concentrations of the fractions were defined abnormal when exceeding two standard deviations of the mean values of 54 normal children reported previously (Siemes et al. 1975). The incidence of abnormal findings in the children of all etiologic groups is summarized in Table II. In all children with C P the relative mobility of the fractions were within normal range. Only in the y3-region additional fractions of oligoclonal yglobulins without a fixed relative mobility appeared in some children. a) Children with CP caused by congenital and postnatal infections
1. Congenital infections Six of the eleven children with congenital infections had cytomegalic inclusion body disease (4 weeks to 18 months of age), three had toxoplasmosis (2'12 to 14 months old) and one child had lues and one rubella, respectively. Fig. 1 shows the CSF-pherogram and its analysis of a child with congenital rubella. N o child of this group had a normal pherogram. There are several remarkable changes: decreased prealbumin in about two thirds of the cases, increased y3-globulin (mainly consisting of I gG) in about 90°/o, an elevated y-trace fraction (y.,)
Vol. 7, No. 3, I976
1
Neuropidiatrie
in ca. three forths and the appearance of oligoclonal yglobulin bands in about one half of the patients. Three of 11 children had an increased az- or ~l-globulin fraction, albumin was lowered in ca. one third of the children. I t should be stressed that only xbout one third of the children had an elevated total protein concentration according to the normal values of different age groups reported by A m m o n and Richterich (1970). 2. Postnatal infections A raised y3-globulin fraction was found in all five children in whom C P was thought to be due to a postnatal infection (8 months to 3 years of age; one child with cytomegalovirus infection, in 4 children a postnatal encephalitis of unknown etiology within the first half-year of life). The 14 months old child with cytomegalic inclusion body disease again showed an oligoclonal protein band in the y-globulin region. A decreased prealbumin was noticed only in one case, whereas two children had an increased prealbumin concentration. A surprising frequent finding was the lowered albumin in three children. b) Children with malformations of CNS
None of the eight children with C P due to malformations ( 3 to 21 months of age) had a normal pherogram. Two of them showed an elevation of total protein and six patients had a lowered prealbumin and an elevated albumin concentration. In 4 children the ysglobulin was increased (see Table 11).
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
Protein patterns of ccrebrospina1 fluid
CP
due to perinatal brain
damage
Only 2 of 16 children with perinatal brain damage showed a normal pherogram. The most obvious abnormaIities were (see Table 11): I . A decrease of prcalbumin or an increase of albumin (about 50 % of the cases) or both (about 40% ). 2. Ambnormal PI-globulin and t-globulin concentrations u p to about 40 % of the children. 3. An clcvated y4-globulin (y-trace) in about 20 5%. Fig. 2 gives an example of a child with CP and hydrocephalus e vacuo due to severe asphyxia. d) Children with CP of unknown etiology
I n this last group of 29 hildren (4 wceks to 6 years of age, see Table 11) 10 had a normal protein profiIe. Again the decrease of prealbumin (ca. 30 %) and increase of albumin (ca. 20 %) were the most obvious changes. Elevation of fll-globulin and lowered r-$0bulin were also found (ca. 15 %). An jnreresting finding seems ro be the
elevation of yJ-globulin (mainly IgG} in 4 children. 11. Relation of protein profile abnormalities to hydrocephalus e vacuo and epilepsy
There was a remarkable association of prealburnin and albumin abnormalities with hydrocephalus e vacuo (see
Table Ill). With exception of the infectious group where in some cases elevated prealbumin andlor decreased albumin values were found hydrocephalus was frequently combined with decreased prealbumin (22 of the 43 cases with hydrocephalus) or increased albumin (20 of the 43 children) or both changes together (15 patients of the 43). Only 3 children without hydrocephalus showed a decreased prealbumin and no child without hydrocephalus had an elevated albumin vaIue. The preal burnin and albumin hanges in the children with hydrocephalus were much more frequent than abnormal total protein concentrations.
Table JII
Incidence of abnormaI total protein concentration and abnormal reIative concencrations of prealbumin and albumin in 43 chiIdren with CP and hydrocephaIus e vacuo
Etiology of
CP
Congenital and posrnatd infections 217 Malformations
318
Pcrinatal brain damage
3!13
Unknown
2/15
317
317 618
2/15
417
317
6/3
6/13
8/13
7/15
6115
1/15
TP = Total Protein, P1 = main Prealbumin fraction, A = Albumin; A, V = increase abovc or decrease below normal range. AP1 + VA, VPI 4-AA = abnormaIities of Prealbumin and Albumin combined, note rhe divergent changes in the infccrious group compared with the others
Downloaded by: National University of Singapore. Copyrighted material.
c) Children with
The protein profiles of children with epilepsy showed no abnormalities that differed horn the ocher patients. Discussion In the present investigation an improved method of a quantitative agarose-geI micro-electropharesjs was applied t o children with CP. Only a few studies on CSF changes in children with CP have been published. The comparison of our results with those of the literature is difficult as the tehniques used for protein electrophoresis vary considerably. Furthermore the age of the patients at the time of lumbar puncture is not documented in most of the publications. Using paper electrophoresis Ha beck (1961) found in 4 out of 9 patients with perinatal brain damage an elevation of (3-globulins and in one case an increased y-globulin. As lowered total protein had been the criteria for sampling the patients the observed abnormalities could be secundary t o this. The CSF of 25 cases with perinatal encephalopathies had been examined by Lowenshal(1964) using agar as well as paper electrophoresis. In patients without epilepsy he observed a certain increase in a-globulins, whereas the normality of fi-globulin vaIues was emphasized. He also noticed that the y-globulin concentrations were normal. In encephalopathies with epilepsy the results were similar, however, the increase of a-globulins was pointed out. Lowenthal came t o the conclusion that the results in perinatal encephaIopathies were normal except in the a-globulins. Bergleiter et al. (1967) reported that
Vol. 7, No. 3, 1916
1
Neuropjdirtric
the paper pherograms in 6 out of 10 paticncs with a pesinatal brain trauma showed increased 8-globulin concentrations. In addition 12 out of 15 patients with cerebral atrophy (demonstrated by pneumencephalography) showed elevated P-gIobulins. Bokon jic and Renders (1964) could not find any abnormalities using agar pherograms in 5 cases with perinatal encephalopathies. Schmidt investigated 90 persons with perinatal trauma by agar-gel electro~horesis. There was no significant change of the CSF protein profile. Not unexpected from the varying results in the literature we found no characteristic pattern for CP. HOWever, one should expect that the CSF protein changes in hildren with CP depend on the cause of thc disorder. By forming groups according to etiology there were several remarkable findings: 1. In children with CP due to congenital and postnatal infections there was a high incidence of elevated yglobulins, particularly 05 the W-globuIin, consisting mainly of Ig G, and the appearance of oligoclonal y-globulin bands.
In infectious diseases of the CNS long standing quantitative and qualitative abnormalities of the y-globulins can not bc attributed ro an increased permeability of the blood-CSF barrier and arc caused by local antibody production within the CNS (Frick and Scheid-Seydel 1958, Cwtler et al. 1970). The discrete antibody bands on the diffuse background of the yJ-gIobulin region are probably caused by restricted
Downloaded by: National University of Singapore. Copyrighted material.
Siemes e t aj.
Protein patterns of cerebrospinal fluid
In our cases this probably characteristic abnormality could be demonstrated up to 18 months of age when cell cound and total protein concentration were normal. 2. In some children with malformations and others with C P of unknown origin the y3-globulin concentration was elevated. This might suggest that an undiagnosed congenital infection was the cause of CP. As the proteins of the y-trace (y,) fraction do not belong to the immunoglobulins the significance of their elevation in some children remains uncertain. 3. Decreased prealbumin andlor increased albumin values were combined with hydrocephalus in about one half of the cases. Compared with ventricular and cisternal fluid spinal subarachnoid fluid of normal individuals shows a lower prealbumin and a higher albu-
min concentration based on regional differences of CSF circulation along the neuraxis. There is a constant protein exchange between the CSF and blood in the absence of local CSF formation in spinal subarachnoid space (Hochwald 1970) which permits albumin to accumulate and s real bum in to decrease. A protein pattern with abnormally decreased prealbumin and increased albumin concentrations can frequently be noticed below an obstructive lesion of the CSF pathway, particularly in spinal subarachnoid space, caused by the persistence of normal protein diffusion while the normal CSF flow is reduced (Matiar-Vahar 1968). According to this observation the similar protein profile in our children with hydrocephalus due to cerebral atrophy may point to a prolonged protein exchange between the CSF and blood due to the enlargement of the CSF pathways with slowed drainage of CSF. The observation of slowed CSF turnover rates demonstrated in adults with normal pressure hydrocephalus would support this hypothesis (Lorenco et a]. 1974). The observations of elevated prealbumin combined with lowered albumin in some children with postencephalitic hydrocephalus points to a different type of CSF hydrodynamics not yet understood. As there is in addition an elevated y3-globulin concentration only in these cases the CSF phorogram indicates the inflammatory origin of hydrocephalus. 4. Apart from the abnormalities of
Downloaded by: National University of Singapore. Copyrighted material.
heterogeneity of the immune response. In the children with congenital infections only a few clones for immunoglobulin production seem to be available as a consequence of immunological immaturity, but the nature of the antigens may be another factor involved (Heremans and Masson 1973). Changes of the electrophoretic protein profile of the CSF in children with congenital infections have not been studied in detail. Only a few cases a t different ages post partum have been reported but in all these children a raised y-globulin concentration was noticed (Arnold and Zetterstrom 1958, Mac/7etanz and H a b e h 1958, Laterre 1965, Siemes 1974).
the prealbumin, albumin and y-globulin concentrations changes of 0,- and 2globulin fractions were noticed in all etiologic groups. The PI-globulin fraction consists mainly of serum transferrin and the 2fraction is made up of a neuraminic acid-deficient transferrin, not found in the serum (Pette and Stupp 1960, Laterre 1965). Frick and Scheid-Seydel (1963) studied intravenously-injected labelled serum transferrin in patients with different diseases of the CNS. Their data indicated that some of the CSF-transferrin was derived immediately from the serum but that an non-labelled pool of C N S was contributing to the CSFtransferrin. When the transferrin concentration was elevated or decreased this seemed to be related to the CNStransferrin pool and the authors postulated an C N S dependant transferrin metabolism. In the 69 children with C P a decrease of the 0,-fraction was found in 8 cases and a lowered 2-fraction in 16 children, 5 of these cases showed both abnormalities combined. As in 9 children there was an association with increased albumin values the lowering of these fractions could be secondary in part of the children. A remarkable change of the P-globulins was a rise of the (31-globulin fraction in 12 of all children with C P (elevation of the z-globulin only in 6 cases). O u r knowledge of the diagnostic significance of elevated P-globulins is rather small, but already in one of the first publications dealing with electro-
Vo1.7, No. 3, 1916
1
Ncuropsdiatrie
phoresis of the CSF one can find the remark that the increase of the PIglobulin points t o a "degenerative" process of brain tissue (Esser and Heinzler 1952). Subsequently this abnormality was found in patients with brain atrophy of different origin, e. g. in cases due to infections and due to brain trauma (Bauer 1953, Bergleiter et al. 1967, Delank 1965, Matiar 1960, Steger 1953). According to these findings one would expect in our study a frequent association of increased 01globulins with hydrocephalus e vacuo, but we found that only 5 out of 12 children with elevated 0,-globulin values had hydrocephalus what meant no coincidence since 43 of the 69 investigated children had hydrocephalus. Machetanz and Habeck (1961) pointed out that an elevation of the PI-globulin concentration was characteristic of the CSF in children suffering from congenital cardiac defects with marked cyanosis, in cases without cyanosis the pherogram was normal.The autors concluded that the PI-globulin increase was due to a "degenerative" process caused by cerebral hypoxia. As no etiologic group was characterized by a decrease or a n increase of one of both S-globulin fractions the meaning of these changes remains unknown. The abnormalities of the y-globulins described in children with CP indicate that electrophoresis of the CSF proteins by an improved technique could be of additional help in diagnosing the origin of this disorder so frequently observed in children.
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
Protein patterns of cerebrospir~alfluid
Zusammenfassung Lumballiquor von 69 Kindern rnit infantiler Zerebralparese verschiedener Genese wurde mit einer verbesserten Methode der Agarosegel-Elektrophorese untersucht. Das Proteinmuster war nur in 12 Fallen normal. Bei Kindern mit einer Zerebralparese infolge einer kongenitalen oder postnatalen Infektion waren erhohte y-Globulinfraktionen und das Auftreten oligoklonaler y-Globulinbanden die bemerkenswertesten Abweichungen. Bei Patienten, bei denen die Zerebralparese durch eine Mii3bildung oder eine perinatale Hirnschadigung und bei Kindern mit einer Zerebralparese unklarer Genese wurden in etwa der Halfte der Falle erniedrigte Praalbumin- und erhohte Albuminwerte in Verbindung mit einem Hydrocephalus e vacuo gefunden. Weniger haufig kamen Veranderungen der 6-Globuline vor, dabei war eine Erhohung der PI-Fraktion seltener als eine Erniedrigung der t-Fraktion. References 1. Ammon, R., Richterich, R.: Die Ermittlung von Normalwerten der Konzentration von Glukose, Protein und Zellen im Liquor des Kindes. Schweiz. med. Wschr. 100: 1317-1320 (1970). 2. Arnold, K. G., Zetterstrom, R.: Proteins in the Cerebrospinal Fluid in the Newborn. Pediatrics 21: 279-387 (1958). 3. Bauer, H . : Uber die Bedeutung der Papier-Elektrophorese des Liquors fiir die klinische Forschung. Dtsch. Z. Nervenheilk. 170: 381-401 (1953).
4. Bergleiter, R., Consbruch, U., Koufen, H . :
Liquor-Beta-Globuline und Hirnatrophie. Arch. Psychiat. Neurol. 210: 140-151 (1967). 5. Bokonjic, R., Renders, J.: Etude electrophorktique de 400 liquides ckphalo-radlidiens pathologiques. Acta Neurol. Belg. 64: 769-776 (1964). 6. Cutler, R. W. P., Watters, G. V., Hammerstadt, J. P.: The origin and turnover rates of cerebrospinal fluid albumin and y-globulin in man. J. neurol. Sci. 10: 259-262 (1970). 7. Delank, H . W.: Das EiweiRbild des Liquor cerebrospinalis und seine klinische Bedeutung. Darmstadt: Steinkopff, 1965. 8. Esser, H., Heinzler, F.: E l e k t r ~ ~ h o r e t i sche EiweiRanalysen im Liquor cerebrospinalis. Dtsch. med. Wschr. 77: 13291330 (1952). 9. Frick, E., Schcid-Seydel, L.: Untersuchungen mit J13'-markiertem Transferrin zur Frage der Abstammung der Liquoreiweif3korper. Klin. Wschr. 41: 589-593 (1963). 10. Frick, E. und Scheid-Seydel, L.: Untersuhungen mit J's1-markicrtem y-Globulin zur Frage der Abstammung der LiquoreiweiRk6rper. Klin. Wschr. 36: 66-69 (1958). 11. Habeck, D.: Zur Verminderung des GesarnteiweiR-Gehaltes im Liquor cerebrospinalis. Arch. Psychiat. Neurol. 202: 354 -370 (1961). 12. Hewmans, J. F. and Masson, P. L.: Specific Analysis of Immunoglobulins. Techniques and Clinical Value. Ciin. Chem. 19: 294-300 (1973). 13. Hochwald, G. M.: Influx of Serum Proteins and their Concentrations in Spinal Fluid along the Neuraxis. J. neurol. Sci. 10: 269-278 (1970). 14. Laterre, E. C.: Lcs protkines du liquide ckphalo-rachidien i l'etat normal et pathologique. Bruxelles: Editions Arscia S. A. 1965. 15. Lorcnco, A. V., Bresnan, M. J., Barlow, Ch. F.: Cerebrospinal Fluid Absorption Deficit in Normal Pressure Hydrocephalus. Arch. Neurol. 30: 387-393 (1974). 16. I.owentha1, A.: Agar Gel Electrophoresis in Neurology. Arnsterdani/New York: Elsevier Publishing Company 1964. 17. Machetanz, E., Habeck, D.: Liquorelektrophoretischc Untersuchungen bei neurologi-
Downloaded by: National University of Singapore. Copyrighted material.
Moreover in part of the children changes of the prealbumin and albumin fractions point t o deviations of the CSF flow caused by hydrocephalus e vacuo.
IS.
19.
20.
21.
22.
schen Erkrankungen im Kindesalter. Zeitschr. Kinderheilk. 81 : 454-471 (1958). Machetanz, E., Habeck, D.: 1st die Hypouie des Zentralnervensystems ein ursachlicher Faktor fiir die p-Globulinvermehrung im Liquor? Arch. Psychiat. Nervenkr. 201: 418-425 (1961). hlatiar, H . : Die Erhohung der p-Globuline irn Liquor. Dtsch. Z. Nervenheilk. 180: 191-215 (1960). Matiar-Vahar, H.: Der lumbale Sperrliquor beim partiellen und kompletten Block. Fortschr. Neurol. Psychiat. 36: 247-257 (1968). Pette, D., Stupp, J.: Die z-Fraktion im Liquor cerebrospinalis. Klin. Wschr. 38: 109-110 (1960). Schmidt, M.: Liquoragargelelektrophorese
Vol. 7, No. 3, 1976
23.
24.
25.
26.
1
Neuropidiatrie
in der neurologisch-psychiatrischen Diagnostik. Jena: VEB Gustav Fischer Verlag 1972. Siegert, M., Siemes, H.: Agarose Gel Electrophoresis of the Proteins of Cerebrospinal Fluid and Analysis of Pherogram Profiles by an Analog Computer. T o be published. Siemes, H., Siegert, M., Lison, H.: Liquorelektrophorese bei connataler Encephalitis. Mschr. Kinderheilk. 122: 440-442 (1974). Siemes, H., Siegert, M. and Rating, D.: Das Liquorproteinprofil normaler Kinder und seine Abhangigkeit vom Lebensalter. Neuropadiatrie 6: 383-397 (1975). Steger, J.: Elektrophoretischc Untersuchungen des Liquors. Dtsch. Z. Nervenheilk. 171: 1-19 (1953).
Downloaded by: National University of Singapore. Copyrighted material.
Siemes et al.
