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Electrophoretic studies of central nervous system proteins
EXPERIMENTAL
NEUROLOGY
Electrophoretk A.
LOWENTHAL,
1, 233-247
Studies D.
(1959)
of Central Proteins1
Nervous M.
KARCHER,AND
VAN
Biochemical Laboratory, Neurological Service, Znstitut Bunge, Laboratory of Chemistry, Institute for Tropical Medicine, Received
March
System
SANDER Berchem-Antwerp, Antwerp, Belgium
and
4, 1959
Paper electrophoresis of cerebrcspinal fluid proteins has demonstrated that the content in gamma globulins may be elevated in certain inflammatory reactions of the central nervous system; it has also demonstrated that certain qualitative differences may exist in the gamma globulin fractions in different cases. We have pursued this line of investigation by biochemical determination cf glycoproteins as well as by efectrophoresis. These preliminary results led us then to attempt to study more specificaffy the qualitative differences, Agar electrophoresis reveals a more detailed subdivision of cerehrospinal fluid proteins into a larger number of phases, and also demonstrates the presence of several phases within the gamma globulin fraction. With the same technique, we have also been able to study the water-soluble proteins of cerebral tissue. Comparison between the electrophoretic patterns obtained on agar from cerebrospinal fluid and from cerebral tissue reveals a number of differences and suggests that certain pathologic gamma globulins would seem to appear at the same time in the spinal fluid and in the brain. It is thus possible to speculate about the endomeningeal Additional studies ccmparing agar electroorigin of those protein fractions. phoretic patterns obtained from human and from animai cerebral tissues reveals an essentially similar pattern.
Introduction
Shortly after the spinal tap was introduced as a diagnostic measurein the clinical diagnosis of neurologic disease,physicians began to realize that in addition to quantitative changesin the proteins contained in the spinal fluid, there were also qualitative alterations. A number of rather crude flocculation tests were only recently replaced by the more exact electrophoretic methods. Paper electrophoresis, supplanting the more 1 Translated from the French by Charles M. Pcser, M.D., University of Kansas School of Medicine. 2 Aided in part by grant No. 108-2 from the National Multiple SclercsIs Society (U. S. A.). The authors wish to thank Dr. Lucien Appel, Institut Bunge, Antwerp, fcr the photographic illustrations. 233
234
LOWENTHAL,
KARCHER,
AND
VAN
tedious Tiselius method, has produced a great the composition of spinal fluid proteins. Since 1952, paper electrophoretic examination an almost routine procedure on our neurologic new method of electrophoretic examination has agar as a substitute for the paper substrate.
SANDE
deal of valuable data on of spinal fluid has become service (8). Recently a been introduced utilizing
Technique
Agar electrophoresis of cerebrospinal fluid is performed using 2 or 3 ml of fluid. The fluid is concentrated by passage through a membrane” under
FIG.
1.
Agar
electrophoretic
pattern
of normal
spinal
fluid;
material
introduced
at arrow.
a pressure of 10 atmospheres (using either air or nitrogen). Electrophoresis is then carried out according to the method of Wieme and Rabaeye (9). After fixation and staining, the color densities are evaluated by means of a special apparatus built by Ressler at the Institute of Tropical Medicine in Antwerp. For electrophoretic examination of cerebral tissue proteins, the same method of electrophoresis is used after homogenization, centrifugation at 20,000 rpm in the cold (0” to ---SO(Z), and extraction with 0.25 M sucrose. Electrophoresis of spinal fluid can be performed only after the fluid is concentrated. Various methods are in current use at the present time. In our laboratory we have found that filtration of the spinal fluid under a pressure of 10 atmospheres through a semipermeable membrane allows full concentration of proteins without qualitative modifications. Using agar instead of paper, concentration must still be performed, but only 2 ml of spinal fluid are required and the migration through agar proceeds more a Ultrafilter
Lsq.
60, Membran-Filter
Gesellschaft,
Gtittingen.
CENTRAL
NERVOUS
SYSTEM
PROTEINS
235
rapidly than on paper (Fig. 1) (2). In general, the quantitative fractionation (Table 1) is similar to that obtained on paper except for the fact that a pre-albumin fraction is more easily identified and can be seen to consist of two or more phases. The beta and gamma globulins can also frequently be demonstrated to contain two or more phases, while albumin and alpha globulins appear to be homogeneous. It is this better detailed fractionation which has allowed us to pursue a more minute study of certain components of the proteins of the central nervous system, both in cerebral tissue itself and in spinal fluid. Results
and
Comments
Paper Electropko~esis of Spinal Fluid. In a large number of human pathologic conditions of the central nervous system, the most frequently encountered modification of the electrophoretic pattern is an increase in the gamma globulin fraction. In some cases, there is elevation of the alpha globulins, whiIe in others there is a reversal of the ratio between alpha-l and alpha-2 globulins (Fig. 2A). In our experience, the latter modification is found with great regularity in spinal fluid obtained from patients with cerebral atrophy secondary to cerebral arteriosclerosis. Pathologic modifications of pre-albumins, albumin, or beta globulins are most unusual. Marked elevation of total protein in spinal fluid, encountered in some patients with brain or high cervical neoplasms, is usually associated with a normal electrophoretic pattern (Fig. 2B). In other situations with high total protein, such as in thoracic or lumbosacral cord compression, the protein fractions are such that the electrophoretic pattern resembles that obtained from serum. Paper Elechophoresis of Spinal Fluid Glycopoteins. In order to investigate the possibility that qualitative differences might exist between fluids showing similar electrophoretic patterns, although obtained from patients with completely different pathologic conditions, we have studied the patterns obtained with stain which would give quantitative values for glycoproteins (4). More specifically, it was felt that the elevation of the gamma globulin fraction in a number of different conditions might represent a qualitative alteration of gamma globulins. We had suspected that there might well be different types of proteins migrating in the fraction usually labeled gamma globulin. The determination of total spinal fluid protein-bound hexoses (4) has indicated that they seem to be closely related to the alpha globulin fraction (Table 2). In some conditions, how-
236
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
CENTRAL
FIG.
sclerosis,
2.
NERVOUS
Paper electrophoretic pattern and B, a case of brain tumor.
SYSTEM
of spinal
PROTEINS
fluid;
A, a case of cerebral
237
arterio-
ever, glycoproteins appeared to be contained in larger quantities in the gamma globulin fraction (Table 3). Thus in certain viral encephalitides and in African trypanosomiasis of the central nervous system there is a definite elevation of the gamma glycoprotein fraction (5). In cases of subacute sclerosing leucoencephalitis, this elevation is either not present or is only of mild degree (Fig. 3). This latter finding may simply be a reflection of the fact that the total spinal fluid protein-bound hexoses content is much lower in subacute sclerosing leucoencephalitis than it is in the other two conditions (Table 4). These observations seemed to confirm our suspicion that there might be qualitative differences in the gamma globulin fraction, since all three conditions are characterized by a striking elevation of the gamma globulin fractions when determined by paper electrophoresis.
LOWENTHAL,
238
RELATIONSHIP
5
10
AND
TABLE 2 GLOBULINS
ALPHA
Percentage of alpha range (Mean k 2a)
Normal 0
BETWEEN
KARCHER,
globulin
15
Concentration
61 -
63 70 -
15
76
37
16
20
78
17
21
-
18 20
28 35 35 37 40 44 56 105 141 -
-
60 68 74
-
HEXOSES~
fluid increased 30
16
23 23 26 27 29 32 35 44 IO 144
AND PROTEIN-BOUND
20 25 of hexoses (mg/liter)
16 -
21
SANDE
in cerebrospinal Significantly
12
-
VAN
a The alpha-globulin concentrations were determined by electrophoresis tein-bound hexoses were determined by chemical analysis of spinal fluid. face figures represent abnormal hexose concentrations, i.e., below or above of mean -C 20. TABLE GLYCOPROTEIN
Source Normal
CSF
Encephalitis Trypanosomiasis Neurosyphilis Subacute sclerosing leucoencephalitis
ELECTROPRORETIC
No. of cases
No. of determinations
17
17
2 5 2 4
35
and proThe bold the range
3 PATTERN
OF SPINAL
FLUID
Globulins Albumin
Alpha-l
Alpha-2
Beta (%)
Gamma (%)
16.6
26.5 (06.3) 29.3 30.0 26.4
10.4 (G2.5) 29.2 43 .o 16.9
14.8
31.4
25.7
(%)
(70)
(%)
18.9 (a4.5)
22.2 (a6.6)
3 5 2
22.0 (a7.0) 13.7 10.6 25.0
15.0
5
15.4
12.7
27.7 16.4
CENTRAL
Frc. 3. Glycoprotein for comparison (lower encephalitis.
NERVOUS
electrophoretic strip) ; A,
pattern subacute
TABLE SPINAL
Source Normal Encephalitis Trypanosomiasis Neurosyphilis Subacute sclerosing leucoencephalitis
FLUID
No. of cases
SYSTEM
239
PROTEINS
(upper sclerosing
strip), and protein leucoencephalitis,
pattern and B,
4
PROTEIN-BOUND
HEXOSES
No. of determinations
Protein-bound
hexoses
(mn %)
3.5 4 6 4
35 6 4
2.07 k 0.54 4.34 9.90 1.80
5
5
2.07
11
Agw EJectrophcwesis of Spinal Fluid Gamm#a Globulins. Agar electrophoresis of spinal fluid has demonstrated the existence of a number of distinct fractions within the gamma globulin phase. This method appears to confirm our preliminary work with the spinal fluid glycoproteins (Fig. 4). In spinal fluid obtained from patients with subacute sclerosing leucoencephalitis, we have been able to separate four distinct phases in the gamma globulin fraction. We have, however, been unable so far to correlate quantitative changes of these different fractions with any clinical entities except to observe marked elevation of some of them in subacute sclerosing leucoencephalitis. Investigation of Origin of Spinal Fluid Protein by Use of ElectroIt has been indicated repeatedly phoresis of Cerebral Tissue Proteins.
240
LOWENTHAL;
FIG. 4. Agar leucoencephalitis;
KARCHER,
electrophoretic pattern material introduced
of spinal at arrow.
AND
fluid
VAN
SANDE
in a case of subacute
sclerosing
that spinal fluid protein probably originates (3)) for the most part, in the serum. This has been done by radioisotope tracing techniques ( 1) as well as by comparing the evolution of spinal fluid and serum proteins in the same patient. This has been well shown in patients with multiple myeloma in whom electrophoretic study of blood, spinal fluid, and urine demonstrate identical abnormal patterns (Fig. 5). The presence of such
FIG.
serum;
5. Protein (upper) and glycoprotein (lower) electrophoretic B, spinal fluid; and C, protein of urine in a case of multiple
patterns myeloma.
of A,
CENTRAL
NERVOUS
SYSTEM
PROTEINS
241
abnormal proteins in the spinal fluid need not be associated with abnormal neurologic signs or symptoms in those patients, nor even by the routine examination of spinal fluid. It has been suggested that an alteration of the blood-spinal fluid barrier allows free passage of such abnormal proteins, possibly in the same manner that occurs in certain infections in which alterations of the alpha and gamma globulins in both serum and spinal fluid can be demonstrated. In other conditions, however, such parallelism cannot be found. In multiple sclerosis the marked modifications of the spinal fluid electrophoretic pattern are not reflected in the serum (6)) while in various encephalitides (5) the evolution of the protein pattern of serum differs markedly from that of the spinal fluid. It is difficult to attempt an explanation for the origin of pre-albumins and especially of certain of the gamma globulins which are not always found in serum, even though they appear in the spinal fluid electrophoretic pattern. These observations inevitably lead to the suggestion that these components, especially some of the gamma globulins, might well originate within the central nervous system itself. Some confirmatory evidence has already been obtained from the study of the glycoproteins of the central nervous system (5). h order to investigate this possibility further, we applied the technique of paper electrophoresis, and later that of agar electrophoresis, to the study of cerebral tissue itself. Paper electrophoretic patterns of cerebral tissue, as a rule, are poorly defined and difficult to interpret (7). In spite of these drawbacks, we felt that there was a definite increase of the gamma globulin fraction in certain structures of the brain of patients with subacute sclerosing leucoencephalitis (Fig. 6). These preliminary
FIG.
acute
6. Paper electrophoretic sclerosing leucoencephalitis
pattern (upper)
of cerebral compared
tissue with
proteins normal
in a case of subbrain (lower).
242
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
results were then followed up by agar electrophoresis. The use of that technique permits fractionation of the proteins in a large number of phases (Table 5). We have noted the presence of two or occasionally three prealbumins while the albumin fraction remains relatively homogeneous; the alpha globulins are comparable to those of the serum and the spinal fluid; the beta globuhns are, in general, divided into two or three phases as are the gamma globulins (Fig. 7). For the sake of convenience, we designated
FIG. 7. introduced
Agar electrophoretic at arrow.
pattern
of norma
cerebral
tissue
proteins;
material
the different phases obtained by this method according to the traditional classification of electrophoretic fractions obtained in serum and spinal fluid. The quantitative results can be summarized as follows: (‘a) The albumin content appears to be smaller in tissue than in either serum or spinal fluid. (b) Pre-albumins are remarkably constant and more abundant in tissue than in spinal fluid. (c) The alpha globulins are relatively more elevated. (d) The composition of the electrophoretic pattern varies according to the anatomic location of the cerebral tissue. The above results represent preliminary observations on normal brains. We have also examined cerebral tissue of patients with subacute sclerosing leucoencephalitis by this method and found a definite augmentation of the gamma globulins (Table 6). Qualitatively, however, these gamma globulins do not appear to be as fragmented as those found in the spinal fluid of the same patients, even though the gamma globulin fraction is equally elevated. This correlation of the quantitative changes of gamma globulin in both cerebral tissue and spinal fluid in patients with subacute sclerosing leucoencephalitis lends considerable support, in our opinion, to the theory of endomeningeal origin of the gamma globulins in this condition.
ganglia
stem
Basal
Brain
Medulla
Cerebellum
matter
CENT
White
Cortex
Region
PER
2.8
8.4 -
4.8 -
-
-
-
-
(1) -
DISTRIBUTION
7.1
31.0
16.7
6.0 8.7
11.3 7.0
13.5
9.6
4.6
8.7 18.7
8.6
12.7 13.8
15.3
12.1
10.0
15.2
14.9
8.0 14.2 9.3
10.0
8.7
7.7 11.5
20.9
15.2
18.6
12.8
7.2
17.1
17.8
18.0
12.7
(2) -
15.9
10.3
10.9
7.3
11.3
TISSUE
Alpha
9.9 7.7
16.7
11.6
(1)
5
25.8
9.1
13.7
9.2
9.0
7.7
(2)
CEREBRAL
TABLE
21.6
34.2
21.8
17.4
21.0
17.8
31.8
9.0
18.5 15.2
21.5
23.5
20.3
27.0
19.3
(1)
Albumin
HUMAN
24.1
IN
26.6 16.6
8.6
1.9
13.6
9.2
(3)
17.5
22.1
10.0
(2)
Pre-albumin
OF PROTEIN
23.5
27.3
15.3 16.6
19.9
23.7 17.5
18.4
22.9
31.5 22.6
19.1
26.2
13.5
30.0
(1)
DETERMINED
34.2
19.0
Beta
4.0
4.9 2.8
4.0
3.6
3.9
4.4
3.9
11.6
4.3 4.3
5.3
3.6
5.5
5.8
(2)
Globulins
BY
ACAR
3.5
2.0
2.7
6.5
6.4
5.6
5.2 4.7
(1)
8.8
5.3
2.7
2.8
3.2
5.5
3.2
-
-
3.5
-
-
2.2 4.5
-
5.7 5.2
7.2
-
-
-
-
(3)
7.4 7.2
46
(2)
10.1
9.0
Gamma
ELECXXOPRORESIS
matter
and
region
a All
Anterior
Gil.
Gray White White
Bod.
matter
cases of subacute
white
matter matter matter
Anterior white matter White matter Posterior white matter Basal ganglia Basal ganglia Corpus callosum Corpus callosum
Gray
Lam.
Case
CENT
20.7
19.5
19.7
-
-
-
sclerosing
7.9
-
leucoencephalitis.
18.9
20.5
14.0 14.8
21.5
-
-
14.4
-
9.7
13.2
8.1
TABLE
22.2
16.7
26.4
20.0
14.7
15.1
24.8
18.0
Albumin
21.0
17.4
10.8
12.1
(2)
OF PROTEIN
(2)
19.3 11.5
23.5
(2)
DISTRIBUTION
21.8
-
(1)
Pre-albumin
PER
6 IN
14.2
9.7
11.7
(1)
21.8 11.6
17.1
12.6 29.6
17.7
13.7
14.6
20.5
Alpha
PATHOLOGIC
8.7
9.8
10.6
(2)
HUMAN
17.4
22.4
25.1
28.2
15.9
15.6
21.6
28.4 36.0
23.4
29.2
25.0
(1)
BRAINS=
Beta
2.6
6.9
5.0 8.1
5.6
4.0
5.0 6.0
7.2
6.2
3.2
3.0
(2)
Globulins
3.9
5.0
3.4
14.0
11.0 14.0
8.0
4.2
5.4
4.6
5.7
7.6
5.0 4.1
6.6
4.4 10.0
5.5
(2)
4.5
(1)
Gamma
z E
s
d
J
E
v! %
6
z
!2
s *
-
-
Monkey Monkey Monkey Monkey Monkey OkaDi
I II III IV V
-
Kangaroo
7.7 7.7
10.5
9.5
9.6
12.7
4.9
8.5
25.3
9.4
15.0
8.1
9.3 14.2
(3)
OF PROTEIN
13.2
(2)
Pre-albumin
DISTRIBUTION
(1)
CENT
Animal
PER
14.3
16.6
21.0
34.0
(1)
IN
22.1
23.3
18.6
28.3
18.0
Albumin
ANIMAL
8.1
19.9
15.0
11.0
(2)
5.0
4.4
24.2
13.1
16.1
14.7 12.4
14.3
8.4
7.3
(2)
TISSUES
Alpha
7
10.8
(1)
CEREBRAL
TABLE
23.2 33.2
26.8
26.7
30.5
27.9
11.3
11.8 38.4
(1)
DETERMINED
AGAR
Beta
4.4
4.5
9.6
9.0
3.9
4.3 3.7
4.2 5.25
(2)
Globulins
BY
3.5 7.2
3.9
2.2
9.0 3.9
8.2 3.9
(1)
2.3
6.2
2.1
4.2
2.1
2.1 0.7
6.0
2.8
(2)
Gamma
ELECTROPRORESIS
_
4.7
-
-
(3)
E g $ m 2 m
z 2
3
2 r” 1: kj
246
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
Additional agar electrophoretic studies of animal cerebral tissue have revealed a composition essentially similar to that in the human (Table 7). This observation parallels results obtained by direct chemical analysis. Conclusions
Electrophoresis of spinal fluid by both paper and agar methods affords a useful approach for clinical investigation of neurologic disease and theoretical study of central nervous system proteins. That qualitative differences may exist in the elevated gamma globulin found in certain pathologic conditions has been confirmed by the use of agar electrophoresis. Different phases, migrating at different speeds, have been identified within the gamma globulin fraction, suggesting that they may well be chemically different. Electrophoretic study of cerebral tissue by means of paper and agar electrophoresis has demonstrated the presence of a different pattern of protein composition as compared to that seen in serum and spinal fluid. Evidence supports the hypothesis that some of the abnormal protein components found in pathologic spinal fluid originate within the central nervous system rather than the serum. No information is available at present concerning the site of origin of these protein fractions within the cerebral parenchyma. References 1. 2.
3. 4. 5. 6.
7.
FRICK, E., and L. SCHEID-SEYDEL, Untersuchungen mit Jrsl-markierten y-globulin. KSn. Wschr. 96, 857-860, 1958. JANSSENS, P., P. CHARLES, M. VAN SANDE, D. KARCHER, and A. LOWENTHAL, Sur C. m-n. la composition du LCR de sujets atteints de trypanosomiase africaine. Sot. biol. 152, 359-362, 1958. KARCHER, D., A. LOWENTHAL, and M. VAN SANDE, L’origine des prottines du LCR. Clin. chim. acta 3, 78-83, 1958. KARCNER, D., M. VAN SANDE, and A. LOWENTHAL, Electropho&se des proteines du LCR (nouvelle technique). Acta din. belg. 12, 538439, 1957. KARCHER, D., M. VAN SANDE, and A. LOWENTHAL, Repartition des glycoprotkines dans le LCR. Rev. belg. path. 26, 325-334, 1958. VAN SANDE, M., D. KARCHER, and A. LOWENTHAL, Examens ClectrophorCtiques des proteines du serum et du liquide cephalo-rachidien chez des patients atteints de sclerose en plaques. Acta new. psychiat. belg. 57, 407-415, 1957. VAN SANWE, M., D. KARCHER, and A. LOWENTHAL, Contribution &ectrophorCtique & I’etude du LCR et du cerveau dans les encephalites aigufs. In “Protides of the Biological Fluids” (Proceedings of the 6th Colloquium, Bruges, 1958). Amsterdam, Elsevier Pub. Co., 1959 (ref. pp. 223-228).
CENTRAL
8.
9.
NERVOUS
SYSTEM
SANDE, M., A. LOWENTHAL, and D. KARCHER, IntCrit phorbigrammes des protkines du liquide cCphalo-rachidien. chiat. belg. 67, 523436, 1957. WIEME, R. G., and M. RABAEYE, A technic of quantitative phoresis. Nututwissenschaften, 6, 112-113, 19.57.
VAN
247
PROTEINS
clinique des klectroActa new. psyultra-micro-electro-
NEUROLOGY
Electrophoretk A.
LOWENTHAL,
1, 233-247
Studies D.
(1959)
of Central Proteins1
Nervous M.
KARCHER,AND
VAN
Biochemical Laboratory, Neurological Service, Znstitut Bunge, Laboratory of Chemistry, Institute for Tropical Medicine, Received
March
System
SANDER Berchem-Antwerp, Antwerp, Belgium
and
4, 1959
Paper electrophoresis of cerebrcspinal fluid proteins has demonstrated that the content in gamma globulins may be elevated in certain inflammatory reactions of the central nervous system; it has also demonstrated that certain qualitative differences may exist in the gamma globulin fractions in different cases. We have pursued this line of investigation by biochemical determination cf glycoproteins as well as by efectrophoresis. These preliminary results led us then to attempt to study more specificaffy the qualitative differences, Agar electrophoresis reveals a more detailed subdivision of cerehrospinal fluid proteins into a larger number of phases, and also demonstrates the presence of several phases within the gamma globulin fraction. With the same technique, we have also been able to study the water-soluble proteins of cerebral tissue. Comparison between the electrophoretic patterns obtained on agar from cerebrospinal fluid and from cerebral tissue reveals a number of differences and suggests that certain pathologic gamma globulins would seem to appear at the same time in the spinal fluid and in the brain. It is thus possible to speculate about the endomeningeal Additional studies ccmparing agar electroorigin of those protein fractions. phoretic patterns obtained from human and from animai cerebral tissues reveals an essentially similar pattern.
Introduction
Shortly after the spinal tap was introduced as a diagnostic measurein the clinical diagnosis of neurologic disease,physicians began to realize that in addition to quantitative changesin the proteins contained in the spinal fluid, there were also qualitative alterations. A number of rather crude flocculation tests were only recently replaced by the more exact electrophoretic methods. Paper electrophoresis, supplanting the more 1 Translated from the French by Charles M. Pcser, M.D., University of Kansas School of Medicine. 2 Aided in part by grant No. 108-2 from the National Multiple SclercsIs Society (U. S. A.). The authors wish to thank Dr. Lucien Appel, Institut Bunge, Antwerp, fcr the photographic illustrations. 233
234
LOWENTHAL,
KARCHER,
AND
VAN
tedious Tiselius method, has produced a great the composition of spinal fluid proteins. Since 1952, paper electrophoretic examination an almost routine procedure on our neurologic new method of electrophoretic examination has agar as a substitute for the paper substrate.
SANDE
deal of valuable data on of spinal fluid has become service (8). Recently a been introduced utilizing
Technique
Agar electrophoresis of cerebrospinal fluid is performed using 2 or 3 ml of fluid. The fluid is concentrated by passage through a membrane” under
FIG.
1.
Agar
electrophoretic
pattern
of normal
spinal
fluid;
material
introduced
at arrow.
a pressure of 10 atmospheres (using either air or nitrogen). Electrophoresis is then carried out according to the method of Wieme and Rabaeye (9). After fixation and staining, the color densities are evaluated by means of a special apparatus built by Ressler at the Institute of Tropical Medicine in Antwerp. For electrophoretic examination of cerebral tissue proteins, the same method of electrophoresis is used after homogenization, centrifugation at 20,000 rpm in the cold (0” to ---SO(Z), and extraction with 0.25 M sucrose. Electrophoresis of spinal fluid can be performed only after the fluid is concentrated. Various methods are in current use at the present time. In our laboratory we have found that filtration of the spinal fluid under a pressure of 10 atmospheres through a semipermeable membrane allows full concentration of proteins without qualitative modifications. Using agar instead of paper, concentration must still be performed, but only 2 ml of spinal fluid are required and the migration through agar proceeds more a Ultrafilter
Lsq.
60, Membran-Filter
Gesellschaft,
Gtittingen.
CENTRAL
NERVOUS
SYSTEM
PROTEINS
235
rapidly than on paper (Fig. 1) (2). In general, the quantitative fractionation (Table 1) is similar to that obtained on paper except for the fact that a pre-albumin fraction is more easily identified and can be seen to consist of two or more phases. The beta and gamma globulins can also frequently be demonstrated to contain two or more phases, while albumin and alpha globulins appear to be homogeneous. It is this better detailed fractionation which has allowed us to pursue a more minute study of certain components of the proteins of the central nervous system, both in cerebral tissue itself and in spinal fluid. Results
and
Comments
Paper Electropko~esis of Spinal Fluid. In a large number of human pathologic conditions of the central nervous system, the most frequently encountered modification of the electrophoretic pattern is an increase in the gamma globulin fraction. In some cases, there is elevation of the alpha globulins, whiIe in others there is a reversal of the ratio between alpha-l and alpha-2 globulins (Fig. 2A). In our experience, the latter modification is found with great regularity in spinal fluid obtained from patients with cerebral atrophy secondary to cerebral arteriosclerosis. Pathologic modifications of pre-albumins, albumin, or beta globulins are most unusual. Marked elevation of total protein in spinal fluid, encountered in some patients with brain or high cervical neoplasms, is usually associated with a normal electrophoretic pattern (Fig. 2B). In other situations with high total protein, such as in thoracic or lumbosacral cord compression, the protein fractions are such that the electrophoretic pattern resembles that obtained from serum. Paper Elechophoresis of Spinal Fluid Glycopoteins. In order to investigate the possibility that qualitative differences might exist between fluids showing similar electrophoretic patterns, although obtained from patients with completely different pathologic conditions, we have studied the patterns obtained with stain which would give quantitative values for glycoproteins (4). More specifically, it was felt that the elevation of the gamma globulin fraction in a number of different conditions might represent a qualitative alteration of gamma globulins. We had suspected that there might well be different types of proteins migrating in the fraction usually labeled gamma globulin. The determination of total spinal fluid protein-bound hexoses (4) has indicated that they seem to be closely related to the alpha globulin fraction (Table 2). In some conditions, how-
236
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
CENTRAL
FIG.
sclerosis,
2.
NERVOUS
Paper electrophoretic pattern and B, a case of brain tumor.
SYSTEM
of spinal
PROTEINS
fluid;
A, a case of cerebral
237
arterio-
ever, glycoproteins appeared to be contained in larger quantities in the gamma globulin fraction (Table 3). Thus in certain viral encephalitides and in African trypanosomiasis of the central nervous system there is a definite elevation of the gamma glycoprotein fraction (5). In cases of subacute sclerosing leucoencephalitis, this elevation is either not present or is only of mild degree (Fig. 3). This latter finding may simply be a reflection of the fact that the total spinal fluid protein-bound hexoses content is much lower in subacute sclerosing leucoencephalitis than it is in the other two conditions (Table 4). These observations seemed to confirm our suspicion that there might be qualitative differences in the gamma globulin fraction, since all three conditions are characterized by a striking elevation of the gamma globulin fractions when determined by paper electrophoresis.
LOWENTHAL,
238
RELATIONSHIP
5
10
AND
TABLE 2 GLOBULINS
ALPHA
Percentage of alpha range (Mean k 2a)
Normal 0
BETWEEN
KARCHER,
globulin
15
Concentration
61 -
63 70 -
15
76
37
16
20
78
17
21
-
18 20
28 35 35 37 40 44 56 105 141 -
-
60 68 74
-
HEXOSES~
fluid increased 30
16
23 23 26 27 29 32 35 44 IO 144
AND PROTEIN-BOUND
20 25 of hexoses (mg/liter)
16 -
21
SANDE
in cerebrospinal Significantly
12
-
VAN
a The alpha-globulin concentrations were determined by electrophoresis tein-bound hexoses were determined by chemical analysis of spinal fluid. face figures represent abnormal hexose concentrations, i.e., below or above of mean -C 20. TABLE GLYCOPROTEIN
Source Normal
CSF
Encephalitis Trypanosomiasis Neurosyphilis Subacute sclerosing leucoencephalitis
ELECTROPRORETIC
No. of cases
No. of determinations
17
17
2 5 2 4
35
and proThe bold the range
3 PATTERN
OF SPINAL
FLUID
Globulins Albumin
Alpha-l
Alpha-2
Beta (%)
Gamma (%)
16.6
26.5 (06.3) 29.3 30.0 26.4
10.4 (G2.5) 29.2 43 .o 16.9
14.8
31.4
25.7
(%)
(70)
(%)
18.9 (a4.5)
22.2 (a6.6)
3 5 2
22.0 (a7.0) 13.7 10.6 25.0
15.0
5
15.4
12.7
27.7 16.4
CENTRAL
Frc. 3. Glycoprotein for comparison (lower encephalitis.
NERVOUS
electrophoretic strip) ; A,
pattern subacute
TABLE SPINAL
Source Normal Encephalitis Trypanosomiasis Neurosyphilis Subacute sclerosing leucoencephalitis
FLUID
No. of cases
SYSTEM
239
PROTEINS
(upper sclerosing
strip), and protein leucoencephalitis,
pattern and B,
4
PROTEIN-BOUND
HEXOSES
No. of determinations
Protein-bound
hexoses
(mn %)
3.5 4 6 4
35 6 4
2.07 k 0.54 4.34 9.90 1.80
5
5
2.07
11
Agw EJectrophcwesis of Spinal Fluid Gamm#a Globulins. Agar electrophoresis of spinal fluid has demonstrated the existence of a number of distinct fractions within the gamma globulin phase. This method appears to confirm our preliminary work with the spinal fluid glycoproteins (Fig. 4). In spinal fluid obtained from patients with subacute sclerosing leucoencephalitis, we have been able to separate four distinct phases in the gamma globulin fraction. We have, however, been unable so far to correlate quantitative changes of these different fractions with any clinical entities except to observe marked elevation of some of them in subacute sclerosing leucoencephalitis. Investigation of Origin of Spinal Fluid Protein by Use of ElectroIt has been indicated repeatedly phoresis of Cerebral Tissue Proteins.
240
LOWENTHAL;
FIG. 4. Agar leucoencephalitis;
KARCHER,
electrophoretic pattern material introduced
of spinal at arrow.
AND
fluid
VAN
SANDE
in a case of subacute
sclerosing
that spinal fluid protein probably originates (3)) for the most part, in the serum. This has been done by radioisotope tracing techniques ( 1) as well as by comparing the evolution of spinal fluid and serum proteins in the same patient. This has been well shown in patients with multiple myeloma in whom electrophoretic study of blood, spinal fluid, and urine demonstrate identical abnormal patterns (Fig. 5). The presence of such
FIG.
serum;
5. Protein (upper) and glycoprotein (lower) electrophoretic B, spinal fluid; and C, protein of urine in a case of multiple
patterns myeloma.
of A,
CENTRAL
NERVOUS
SYSTEM
PROTEINS
241
abnormal proteins in the spinal fluid need not be associated with abnormal neurologic signs or symptoms in those patients, nor even by the routine examination of spinal fluid. It has been suggested that an alteration of the blood-spinal fluid barrier allows free passage of such abnormal proteins, possibly in the same manner that occurs in certain infections in which alterations of the alpha and gamma globulins in both serum and spinal fluid can be demonstrated. In other conditions, however, such parallelism cannot be found. In multiple sclerosis the marked modifications of the spinal fluid electrophoretic pattern are not reflected in the serum (6)) while in various encephalitides (5) the evolution of the protein pattern of serum differs markedly from that of the spinal fluid. It is difficult to attempt an explanation for the origin of pre-albumins and especially of certain of the gamma globulins which are not always found in serum, even though they appear in the spinal fluid electrophoretic pattern. These observations inevitably lead to the suggestion that these components, especially some of the gamma globulins, might well originate within the central nervous system itself. Some confirmatory evidence has already been obtained from the study of the glycoproteins of the central nervous system (5). h order to investigate this possibility further, we applied the technique of paper electrophoresis, and later that of agar electrophoresis, to the study of cerebral tissue itself. Paper electrophoretic patterns of cerebral tissue, as a rule, are poorly defined and difficult to interpret (7). In spite of these drawbacks, we felt that there was a definite increase of the gamma globulin fraction in certain structures of the brain of patients with subacute sclerosing leucoencephalitis (Fig. 6). These preliminary
FIG.
acute
6. Paper electrophoretic sclerosing leucoencephalitis
pattern (upper)
of cerebral compared
tissue with
proteins normal
in a case of subbrain (lower).
242
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
results were then followed up by agar electrophoresis. The use of that technique permits fractionation of the proteins in a large number of phases (Table 5). We have noted the presence of two or occasionally three prealbumins while the albumin fraction remains relatively homogeneous; the alpha globulins are comparable to those of the serum and the spinal fluid; the beta globuhns are, in general, divided into two or three phases as are the gamma globulins (Fig. 7). For the sake of convenience, we designated
FIG. 7. introduced
Agar electrophoretic at arrow.
pattern
of norma
cerebral
tissue
proteins;
material
the different phases obtained by this method according to the traditional classification of electrophoretic fractions obtained in serum and spinal fluid. The quantitative results can be summarized as follows: (‘a) The albumin content appears to be smaller in tissue than in either serum or spinal fluid. (b) Pre-albumins are remarkably constant and more abundant in tissue than in spinal fluid. (c) The alpha globulins are relatively more elevated. (d) The composition of the electrophoretic pattern varies according to the anatomic location of the cerebral tissue. The above results represent preliminary observations on normal brains. We have also examined cerebral tissue of patients with subacute sclerosing leucoencephalitis by this method and found a definite augmentation of the gamma globulins (Table 6). Qualitatively, however, these gamma globulins do not appear to be as fragmented as those found in the spinal fluid of the same patients, even though the gamma globulin fraction is equally elevated. This correlation of the quantitative changes of gamma globulin in both cerebral tissue and spinal fluid in patients with subacute sclerosing leucoencephalitis lends considerable support, in our opinion, to the theory of endomeningeal origin of the gamma globulins in this condition.
ganglia
stem
Basal
Brain
Medulla
Cerebellum
matter
CENT
White
Cortex
Region
PER
2.8
8.4 -
4.8 -
-
-
-
-
(1) -
DISTRIBUTION
7.1
31.0
16.7
6.0 8.7
11.3 7.0
13.5
9.6
4.6
8.7 18.7
8.6
12.7 13.8
15.3
12.1
10.0
15.2
14.9
8.0 14.2 9.3
10.0
8.7
7.7 11.5
20.9
15.2
18.6
12.8
7.2
17.1
17.8
18.0
12.7
(2) -
15.9
10.3
10.9
7.3
11.3
TISSUE
Alpha
9.9 7.7
16.7
11.6
(1)
5
25.8
9.1
13.7
9.2
9.0
7.7
(2)
CEREBRAL
TABLE
21.6
34.2
21.8
17.4
21.0
17.8
31.8
9.0
18.5 15.2
21.5
23.5
20.3
27.0
19.3
(1)
Albumin
HUMAN
24.1
IN
26.6 16.6
8.6
1.9
13.6
9.2
(3)
17.5
22.1
10.0
(2)
Pre-albumin
OF PROTEIN
23.5
27.3
15.3 16.6
19.9
23.7 17.5
18.4
22.9
31.5 22.6
19.1
26.2
13.5
30.0
(1)
DETERMINED
34.2
19.0
Beta
4.0
4.9 2.8
4.0
3.6
3.9
4.4
3.9
11.6
4.3 4.3
5.3
3.6
5.5
5.8
(2)
Globulins
BY
ACAR
3.5
2.0
2.7
6.5
6.4
5.6
5.2 4.7
(1)
8.8
5.3
2.7
2.8
3.2
5.5
3.2
-
-
3.5
-
-
2.2 4.5
-
5.7 5.2
7.2
-
-
-
-
(3)
7.4 7.2
46
(2)
10.1
9.0
Gamma
ELECXXOPRORESIS
matter
and
region
a All
Anterior
Gil.
Gray White White
Bod.
matter
cases of subacute
white
matter matter matter
Anterior white matter White matter Posterior white matter Basal ganglia Basal ganglia Corpus callosum Corpus callosum
Gray
Lam.
Case
CENT
20.7
19.5
19.7
-
-
-
sclerosing
7.9
-
leucoencephalitis.
18.9
20.5
14.0 14.8
21.5
-
-
14.4
-
9.7
13.2
8.1
TABLE
22.2
16.7
26.4
20.0
14.7
15.1
24.8
18.0
Albumin
21.0
17.4
10.8
12.1
(2)
OF PROTEIN
(2)
19.3 11.5
23.5
(2)
DISTRIBUTION
21.8
-
(1)
Pre-albumin
PER
6 IN
14.2
9.7
11.7
(1)
21.8 11.6
17.1
12.6 29.6
17.7
13.7
14.6
20.5
Alpha
PATHOLOGIC
8.7
9.8
10.6
(2)
HUMAN
17.4
22.4
25.1
28.2
15.9
15.6
21.6
28.4 36.0
23.4
29.2
25.0
(1)
BRAINS=
Beta
2.6
6.9
5.0 8.1
5.6
4.0
5.0 6.0
7.2
6.2
3.2
3.0
(2)
Globulins
3.9
5.0
3.4
14.0
11.0 14.0
8.0
4.2
5.4
4.6
5.7
7.6
5.0 4.1
6.6
4.4 10.0
5.5
(2)
4.5
(1)
Gamma
z E
s
d
J
E
v! %
6
z
!2
s *
-
-
Monkey Monkey Monkey Monkey Monkey OkaDi
I II III IV V
-
Kangaroo
7.7 7.7
10.5
9.5
9.6
12.7
4.9
8.5
25.3
9.4
15.0
8.1
9.3 14.2
(3)
OF PROTEIN
13.2
(2)
Pre-albumin
DISTRIBUTION
(1)
CENT
Animal
PER
14.3
16.6
21.0
34.0
(1)
IN
22.1
23.3
18.6
28.3
18.0
Albumin
ANIMAL
8.1
19.9
15.0
11.0
(2)
5.0
4.4
24.2
13.1
16.1
14.7 12.4
14.3
8.4
7.3
(2)
TISSUES
Alpha
7
10.8
(1)
CEREBRAL
TABLE
23.2 33.2
26.8
26.7
30.5
27.9
11.3
11.8 38.4
(1)
DETERMINED
AGAR
Beta
4.4
4.5
9.6
9.0
3.9
4.3 3.7
4.2 5.25
(2)
Globulins
BY
3.5 7.2
3.9
2.2
9.0 3.9
8.2 3.9
(1)
2.3
6.2
2.1
4.2
2.1
2.1 0.7
6.0
2.8
(2)
Gamma
ELECTROPRORESIS
_
4.7
-
-
(3)
E g $ m 2 m
z 2
3
2 r” 1: kj
246
LOWENTHAL,
KARCHER,
AND
VAN
SANDE
Additional agar electrophoretic studies of animal cerebral tissue have revealed a composition essentially similar to that in the human (Table 7). This observation parallels results obtained by direct chemical analysis. Conclusions
Electrophoresis of spinal fluid by both paper and agar methods affords a useful approach for clinical investigation of neurologic disease and theoretical study of central nervous system proteins. That qualitative differences may exist in the elevated gamma globulin found in certain pathologic conditions has been confirmed by the use of agar electrophoresis. Different phases, migrating at different speeds, have been identified within the gamma globulin fraction, suggesting that they may well be chemically different. Electrophoretic study of cerebral tissue by means of paper and agar electrophoresis has demonstrated the presence of a different pattern of protein composition as compared to that seen in serum and spinal fluid. Evidence supports the hypothesis that some of the abnormal protein components found in pathologic spinal fluid originate within the central nervous system rather than the serum. No information is available at present concerning the site of origin of these protein fractions within the cerebral parenchyma. References 1. 2.
3. 4. 5. 6.
7.
FRICK, E., and L. SCHEID-SEYDEL, Untersuchungen mit Jrsl-markierten y-globulin. KSn. Wschr. 96, 857-860, 1958. JANSSENS, P., P. CHARLES, M. VAN SANDE, D. KARCHER, and A. LOWENTHAL, Sur C. m-n. la composition du LCR de sujets atteints de trypanosomiase africaine. Sot. biol. 152, 359-362, 1958. KARCHER, D., A. LOWENTHAL, and M. VAN SANDE, L’origine des prottines du LCR. Clin. chim. acta 3, 78-83, 1958. KARCNER, D., M. VAN SANDE, and A. LOWENTHAL, Electropho&se des proteines du LCR (nouvelle technique). Acta din. belg. 12, 538439, 1957. KARCHER, D., M. VAN SANDE, and A. LOWENTHAL, Repartition des glycoprotkines dans le LCR. Rev. belg. path. 26, 325-334, 1958. VAN SANDE, M., D. KARCHER, and A. LOWENTHAL, Examens ClectrophorCtiques des proteines du serum et du liquide cephalo-rachidien chez des patients atteints de sclerose en plaques. Acta new. psychiat. belg. 57, 407-415, 1957. VAN SANWE, M., D. KARCHER, and A. LOWENTHAL, Contribution &ectrophorCtique & I’etude du LCR et du cerveau dans les encephalites aigufs. In “Protides of the Biological Fluids” (Proceedings of the 6th Colloquium, Bruges, 1958). Amsterdam, Elsevier Pub. Co., 1959 (ref. pp. 223-228).
CENTRAL
8.
9.
NERVOUS
SYSTEM
SANDE, M., A. LOWENTHAL, and D. KARCHER, IntCrit phorbigrammes des protkines du liquide cCphalo-rachidien. chiat. belg. 67, 523436, 1957. WIEME, R. G., and M. RABAEYE, A technic of quantitative phoresis. Nututwissenschaften, 6, 112-113, 19.57.
VAN
247
PROTEINS
clinique des klectroActa new. psyultra-micro-electro-