Isoelectric focusing of CSF proteins in neurological diseases

Isoelectric focusing of CSF proteins in neurological diseases

Journal of the neurological Sciences, 1974, 23:19%213 199 © Elsevier Scientific Publishing Company, Amsterd~tm - Printed in The Netherlands Isoelec...

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Journal of the neurological Sciences, 1974, 23:19%213

199

© Elsevier Scientific Publishing Company, Amsterd~tm - Printed in The Netherlands

Isoelectric Focusing of CSF Proteins in Neurological Diseases K. G. KJELLIN AND O. VESTERBERG Department of Neurology (K.G.K.), Karolinska Hospital, S-104 O1 Stockholm 60, and Chemical Division (0. V.), Occupational Health Department, The National Board of Occupational Safety and Health, S-IO0 26 Stockholm 34 (Sweden) (Received 1 March, 1974)

INTRODUCTION

Analysis of the cerebrospinal fluid (CSF) proteins is usually carried out by agar or paper electrophoresis, or sometimes by immunoelectrophoresis while other techniques have been used less frequently (Lowenthal 1964; Clausen, Fog and Einstein 1%9). The abnormal CSF-protein signs found by quantitative paper electrophoresis can be classified according to characteristic "patterns", or their probable combinations, after correction for the estimated influence of the serum protein distribution, as described by Kjellin (1969) and G~trde and Kjellin (1971): the "BCDI" system where "B" means barrier damage, "C" CSF block, "D" "degenerative" pattern, and "I .... immunopathic" pattern. (The characteristic feature of the "immunopathic" pattern is a relative increase of the CSF gamma-globulins which is higher than might be expected from the serum gamma-globulin concentration at the same time and any existing barrier damage, e.g. indicating gamma-globulin production within the CNS and/or meninges.) Isoelectric focusing is a new technique with high resolving capacity by which proteins are separated according to their isoelectric points. The method was made practicable by the introduction of carrier ampholytes, and proteins with such small differences in their isoelectric points as 0.01-0.02 can be separated from each other (Vesterberg and Svensson 1966). The very high resolving capacity for proteins can even surpass that attainable with disc-electrophoresis (Drawert and G6rg 1972; Vesterberg 1973). Some investigators have shown that isoelectric focusing is especially suited for studies of gamma-globulins (Williamson 1971). During recent years the method ofisoelectric focusing has been adapted for analytical studies of many proteins by performing the focusing in polyacrylamide gel. In earlier studies many authors performed the focusing procedure in gel cylinders. However, evidence now accumulating favours the use of flat beds or thin layers of polyacrylamide This investigation was supported by a grant from the Swedish Multiple Sclerosis Fund.

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gel on glass plates. The most important advantages have been summed up by Vestcr.berg (1973, 1974) and Davies (1974). Many samples can be run in parallel side by side. which is helpful for comparative purposes, and samples with very low protein contents (#g quantities) can be examined. Kjellin (1972, 1973a) used different isoelectric focusing techniques in separating xanthochromic CSF-protein compounds, and found the thin layer procedure in polyacrylamide gel to be the most suitable one for clinical examinations. The potential possibilities of isoelectric focusing for the separation of CSF proteins were pointed out by Fossard, Dale and Latner (1970). Moreover, one procedure for thin layer isoelectric focusing in examining CSF proteins in multiple sclerosis (MS) patients was reported by Delmotte (1971, 1972), and another by Kjellin and Vesterberg (1972). In all cases of verified MS which have been studied, high concentrations of CSF gamma-globulins with alkaline isoelectric points were found. Kjellin (1973b) and Kjellin and Vesterberg (1972, 1973) also reported the CSF-protein patterns observed in subjects with other demyelinating and infectious disorders, and Kjellin (1972, 1973a) described the xanthochromic protein patterns found in CSF from patients with haemorrhagic CNS diseases. Preliminary reports on the present investigation have been given by Kjellin and Vesterberg (1972, 1973) and Kjellin (1973b). MATERIAL A N D METHODS

The CSF samples were collected from patients treated in the Department of Neurology, Karolinska Hospital, Stockholm. Besides control subjects with normal CSF-protein findings, the material from patients included predominantly cases with verified or suspected MS, myelopathies, encephalomyelomeningitis and polyneuropathies. The assessment of the clinical data, excluding the CSF signs, was based on close examination of the clinical records and of the records of outpatient follow-up. The CSF was obtained by lumbar puncture; generally about 10 ml were withdrawn at one puncture. Serum samples were collected parallel with the lumbar punctures. Besides isoelectric focusing and quantitative paper electrophoresis, the CSF was examined for cells, protein concentration, and in many cases mastix reaction. The cells in the CSF were examined by routine microscopy and counted per 3.2 mm 3. The CSF-protein concentration was determined by a modification of the method of Lowry, Rosebrough, Farr and Randall (1951). The quantitative paper electrophoretic examinations of CSF and serum were performed as described previously (see Ggtrde and Kjellin 1971). The results of the CSF electrophoresis were classified according to characteristic "patterns", or their probable combinations, after correction for the estimated influence of the serum protein distribution (Kjellin 1969), and the CSF patterns were classified according to Ggtrde and Kjellin (1971). The CSF samples were ultrafiltered (see G~trde and Kjellin 1971 ), and concentrated about 10 times, at which stage samples were withdrawn for isoelectric focusing, and the samples were then further concentrated to about 100 times for quantitative paper

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electrophoresis. The amounts (20-40 pl) of the concentrated CSF used for isoelectric focusing, were selected in order to get similar protein contents in all CSF samples examined. A serum sample diluted with physiological NaC1 solution 10 times, from the same patient, was applied side by side with the corresponding concentrated CSF sample on the acrylamide gel. The isoelectric focusing procedure was performed in polyacrylamide gel in principle as described by Vesterberg (1972, 1973), the pH range in the gel usually being 3-11:6 ml of a water solution of acrylamide (British Drug House) 30.5 ~o, and of bisacrylamide 1.0 ~o, 22 ml aqua dest. and 3.75 g sucrose. Several Ampholine solution mixtures were tested, and for separating CSF proteins in the pH range 3.5-11 the two following were preferred: Ampholine solutions (LKB) pH 3.5-10 (1.4 ml), pH 4-6 (0.1 ml), pH 5-7 (0.1 ml) with pH 9-11 (0.2 o r 0.4 ml). The sample solutions were soaked in pieces of"surgical lint" as described by Vesterberg (1972). The isoelectric focusing was started at 550V, and with 90 mA when 0.2 ml of the Ampholine solution 9-11 was used, but 94 mA when 0.4 ml of the same Ampholine solution was chosen. The isoelectric separation was performed in 60 rain with a final voltage of 1000V (final current 15 mA). For special studies more narrow pH ranges were used, mainly pH 8-10. Details of such methods can be found in a recent paper by Vesterberg (1973). Due to the good cooling efficiency, the total electrical load could be as high as 45W until a final voltage of 1000V was reached. Close to each electrode 2/A of a haemoglobin solution containing 7 mg haemoglobin/ml was applied. The joining of the haemoglobin bands migrating from both electrodes took place within 1 hr. Usually 8 samples, 4 CSF samples and the corresponding 4 serum samples, as well as the haemoglobin standard sample, were focused on each gel slab. Staining of proteins and pH measurements after isoelectric focusing were performed as described by Vesterberg (1972). Recently we have been able to make a densitometric evaluation of the separated proteins by using a Zeiss PMQ2 densitometer with a Program Spectral Photometer (Kjellin and Vesterberg, to be published). RESULTS

Some typical CSF findings in cases examined by isoelectric focusing are shown in Figs. 1-3. The results of the first series of cases, the subject of a preliminary report at the 20th Congress of Scandinavian Neurologists (Kjellin and Vesterberg 1972) are given in Tables 1 and 2. The results of isoelectric focusing and quantitative paper electrophoresis were compared. Twelve cases presumed to have "normal" CSF as judged from cell examinations, protein determinations, and quantitative paper electrophoresis, were used as control cases. The number of such control cases (epilepsy, cephalalgia, psychoneurosis) was later extended to 23 subjects. The isoelectric focusing patterns with increased alkaline fractions (pH > 8) found in all cases with MS were labelled with the tentative working term "MS" in this series. The isoelectric focusing pattern "B" (Table 2) indicated that the CSF gamma-globulin range seemed to be identical

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Fig. 1. Gel isoelectric focusing.pattern of normal CSF. Serum sample from the same patient to the left, and a reference solution of haemoglobin to the right. The anode was at the top and the cathode at the bottom.

TABLE l T H E R E S U L T S IN T H E F I R S T SERIES O F CASES

Reported at the 20th Congress of Scandinavian Neurologists (Kjellin and Vesterberg 1972).

Diaanosis Control cases (12) (cephalalgia, epilepsy, emotional disorders) MS(ll) ?MS (10) Myelopathy of unknown cause (7)

PELF (quantitative paper electrophoresis) nothing abnormal (12)

IF (isoelectric focusing) nothing abnormal (t2)

I (5), BI (6)

"'MS" (1 l)

I (5), I? (1), BI (3), B + I? (1)

" M S " (7), "MS"? (2), "MS"-neg. 0)

I (4), BI (1), B + I ? (2)

" M S " (5), "MS"? (2)

Symbols used: B = b a r r i e r damage; I = " i m m u n o p a t h i c " pattern; " M S " = increased alkaline fractions in the range of pH > 8. Numbers of cases in brackets.

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Fig. 2. Gel isoelectric focusing pattern of barrier damage in the CSF. Serum sample from the same patient to the right. The CSF gamma-globulins (lower half of the pattern) are increased rather uniformly, similar to a "finger-print" of the corresponding serum gamma-globulin pattern (pattern " f " in Table 3). The anode was at the top and the cathode at the bottom. TABLE 2 THE RESULTSOF THE FIRSTSERIESOF CASES Reported at the 20th Congress of Scandinavian Neurologists (Kjellin and Vesterberg 1972) Numbers of cases in brackets. Diagnosis B 12-Neuropathy (2) B lz-Neuropathy, treated (1) Combined neuropathy, st.p. Herpes zoster (1) Cervical myelopathy (2) Chronic meningitis (2) Meningoencephalitis (1) Polyneuropathy alcoholic (1) CMT (1) Symbols used : see Table 1.

PELF (quantitative paper electrophor esis)

IF ( isoelectric focusing)

I (1), I? (1) nothing abnormal (1) B (1)

alkaline fraction at about pH 7.5 (2) nothing abnormal (1) B (1)

B, slight (2) BI (2) B (1)

nothing noteworthy (2) B (1), " M S " (1) " M S " (1)

B (1) nothing noteworthy (1)

B (1) nothing noteworthy (1)

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K. (;. KJELLIN, O. VESTERBERG

~ii2i;i~;i

A

Fig. 3. Gel isoelectric focusing patterns of CSF. A : a typical CSF pattern found in MS. Serum sample from the same patient to the right. The CSF gamma-globulin region extending below the corresponding serum gamma-globulin range, with pronounced increase of alkaline fractions pH > 8. Note also the isolated. most alkaline end-fraction of the CSF, and the double fractions in "region 5"" (see Fig. 5). The latter two findings, indicated by arrows, were frequently observed in MS. B: CSF pattern found in a case of chronic meningoencephalitis. Serum sample from the same patient to the left. The CSF gamma-globulin region extending below the corresponding serum gamma-globulin range, with a pronounced increase of fractions in a broad range. Note the m a n y discrete bands especially in the middle range of the CSF gamma-globulin region. The anode was at the top and the cathode at the bottom.

("finger-print") with the pattern of the corresponding serum sample. In 2 cases of Blz-neuropathy (Table 2), an alkaline fraction at about pH 7.5 was found (Fig. 4), which disappeared in one re-examined case after treatment of the B12-deficiency. A third case of B~2-neuropathy with a similar CSF-fraction on isoelectric focusing was later added. In a second series of cases (Tables 4-7) the CSF-protein fractions obtained by isoelectric focusing were divided into 10 regions, numbered 1-10 from the anodic side (Fig. 5). The different patterns found in the CSF gamma-globulin range were classified in the groups a-f, as shown in Table 3. The isoelectric focusing patterns of CSF gamma-globulins (a-t) observed in pa-

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Fig. 4. Gel isoelectric focusing pattern of CSF from a patient with B12-neuropathy. An alkaline fraction at about pH 7.5 is observed in the gamma-globulin region. The anode was at the top and the cathode at the bottom. TABLE 3 THE ISOELECTRICFOCUSINGPATTERNS(a-f) OF CSF GAMMA-GLOBULINSDIVERGINGFROMTHE "NORMAL" FINDINGSIN 23 CONTROLCASES Visual valuation where the corresponding serum protein patterns have been taken into consideration. Pattern

Relative increase of proteins in the regions 7-10 (see Fig. 5) 8-10, especially 9 10 8-10, rather uniformly 7-10, especially 8-10 8-9, rather uniformly 7-9, especially 9 7-9, rather uniform, usually similar to a "finger-print" of the corresponding serum gamma-globulin pattern

tients w i t h different n e u r o l o g i c a l d i s o r d e r s , h a v e b e e n c o m p a r e d w i t h t h e p a t t e r n s o f q u a n t i t a t i v e p a p e r e l e c t r o p h o r e s i s o f t h e s a m e C S F s a m p l e s ( T a b l e s 4-7). I n 21 p a t i e n t s w i t h c l i n i c a l l y v e r i f i e d M S i s o e l e c t r i c f o c u s i n g p a t t e r n s a - e w e r e

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K. G. KJELLIN, O. VESTERBERG TABLE 4

T H E S E C O N D SERIES OF CASES;

CSF

P R O T E I N P A T T E R N S F O U N D IN C L I N I C A L I . Y V E R n : I E D A N D SUSPE( FED M N

Numbers of cases in brackets. Diagnosis

IF ( isoelectric focusing)

MS (21)

a b c d e

MS? (18)

PELF (quantitative paper electrophoresis )

(4) (2) (8) (4) (3)

1, pronounced (3); BI, moderate (1) I, pronounced (2) I, pronounced (5); BI, moderate (2), slight (1) I, moderate (1), slight (2); BI, moderate (1) L pronounced (1), slight (1); BI, pronounced (1)

a (3) d (10)

I, pronounced (1), slight (1); B, pronounced (1) I, pronounced (2), moderate (1), slight (3); BI, slight (1); 1? (3) I, slight (1); BI, slight (1) I, slight (1): I? (1) nothing noteworthy (1)

e (2) f (2) nothing noteworthy (1) Symbols used : see Table 1 and Table 3.

TABLE5 T H E S E C O N D SERIES OF C A S E S :

CSF

P R O T E I N P A T T E R N S F O U N D I N M Y E L O P A T H Y OF U N K N O W N

AETIOLOGY AND

POLYNEUROPATHY

Numbers of cases in brackets Diagnosis

IF ( isoelectric focusing)

PELF (quantitative paper electrophoresis)

c (3) d (4)

1, slight (1); BI, pronounced (1); B, moderate (1) BI, slight (1); B, pronounced + I, slight (1); CSF beta 1 increased (1); B? (I) B, pronounced ( 1): CS F beta 1 increased (1)

Myelopathy of unknown aetiology (9)

f (2) Polyneuropathy (7) alcoholic (1) diabetic (1) Guillain-Barr6 syndrome ( 1 ) unknown aetiology (4)

f (1) f (1) f ( 1) f (3) nothing abnormal (1)

nothing abnormal (1) B, slight (1) B I, slight ( 1) B, pronounced (2); I? (1) nothing abnormal (1)

Symbols used : see Table 1 and Table 3.

found, while none of the cases showed an "f"-pattern. The "c"-pattern dominated and was observed in nearly 40 ~o of the cases. The quantitative paper electrophoresis of the CSF showed an "immunopathic" pattern ("I"-pattern) in about 70 ~o of the cases, and mixed "barrier damage+immunopathic" pattern ("BI"-pattern) in the remaining cases.

Symbols used : see Table 1 and Table 3.

Hemiplegia of unknown origin (7)

Encephalopathy (undiagnosed) (7)

Tumour, intracranial (4) acoustic neuroma (2) medulloblastoma (1) meningioma (1)

Meningoencephalitis (4)

Diagnosis

c(1) f (6) d(1) f (5) nothing abnormal (1)

f (2) f (1) f (1)

c (3) f (1)

IF ( isoelectric focusing)

PELF (quantitative paper electrophoresis )

not performed B, moderate (1), slight (2); I? (I); nothing abnormal (2) B, slight (I) B, pronounced (1), slight (2); I? (1); nothing abnormal (1) nothing abnormal (1)

B, pronounced (1); BI, moderate (1) B, pronounced (1) prealbumins and beta 1 increased in CSF (I)

BI, pronounced (3) B, moderate (1)

Numbers of cases in brackets.

THE SECOND SERIES OF CASES ~ C Z F PROTEIN PATTERNS FOUND IN MEN1NGOENCEPI-IALITIS~ INTRACRANIAL TUMOUR, ENCEPIJALOPATHY AND HEMIPLEGIA OF U N K N O W N AETIOLOGY

TABLE 6

-.-.-I

Z

7~

2~

(3

(3

m

©

nothing abnormal (1) I, slight (1); nothing abnormal (l) D, moderate (1) B, slight (1); nothing abnormal (1} B, pronounced (1) nothing abnormal (1) nothing abnormal (2) B, moderate (1)

nothing abnormal (1) d (2) f (1) f (2) f (I) nothing abnormal (I) f (1); nothing abnormal (1) d (1) f (2) f (1)

Hereditary ataxia (1) Cranial nerve lesions (10) optic neuritis (2) tobacco amblyopia (1) optic nerve lesion (2) third nerve paralysis (I) sixth nerve paralysis (1) trigeminal neuralgia (2) recurrent facial paralysis (1)

Systemic diseases (3) collagen tissue diseases (2) Hodgkin's disease (1)

Symbols used: see Table 1 and Table 3. "D" ="degenerative disease" pattern.

B, moderate (1); D, slight (1); I, slight (1) nothing abnormal (1)

f (4)

Cerebellar syndromes (4)

B, moderate (1); nothing abnormal (1) nothing abnormal (1)

B, moderate (2) nothing abnormal (l)

PELF (quantitative paper electrophoresis)

f (2) nothing abnormal (1)

IF ( isoelectric Jbcusing)

Amyotrophic lateral sclerosis (3)

Diagnosis

Numbers of cases in brackets.

THE SECONDSERIESOF CASES: CSF PROTEINPATTERNSFOUNDIN SOMEOTHERDISORDERS

TABLE 7

O .<

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Isoelectrlc focusing of CSF protelns: Numbered regions.

Sample application

Sample application

123456 ~, 7 8 9 10 , , , J i , , i i i Acid

<

123,456 J,i , ~ , t ,

~ Alkaline

Acid

7

,

8

(

t

9

,

10

) Alkaline

Sample applcation

Sample application

a.

b.

~

.

.

.

.

.

.

d.

.

ET

II

..

,

c

Fig. 5. The isoelectric focusing patterns (a-f) of CSF gamma-globulins as defined in Table 3. Black and hatched areas indicate relative increase of proteins, where hatched areas are used only to point out a less pronounced increase than in black areas in the patterns a, c and e.

In 18 patients with clinically suspected MS "a"-, "d"-, and "e"-patterns were found in 15 cases, while an "f"-pattern was observed in 2, and nothing noteworthy in the remaining 1. In the last-mentioned case the quantitative paper electrophoresis also showed normal findings. The "d"-pattern was dominant in this group of patients, and clearly-defined in about 55 ~ of the cases. In 9 patients with myelopathy of unknown aetiology (in spite of exhaustive examinations including myelography), "c"- or "d"-patterns were observed in 7 cases, and an "f"-pattern in the other 2. The quantitative paper electrophoresis of the CSF showed "I"- or "BI"-patterns in 4 of the cases, "B"-pattern (definite or suspected) in 3 cases, and increased CSF betat-globulin concentrations in the remaining 2. It may be mentioned that in the 2 cases with an "f"-pattern observed on isoelectric focusing of the CSF, the quantitative paper electrophoresis showed a "B"-pattern and an increased concentration of CSF betat-globulins respectively.

DISCUSSION

The extremely high resolving power of isoelectric focusing for CSF proteins has previously been pointed out by Kjellin and Vesterberg (1972, 1973), one very important feature of the method here being that proteins in the gamma-globulin region can be separated into many fractions as demonstrated in cases of demyelinating, infective and barrier-damage disorders. Another important finding was that the CSF albumin can be subdivided into 2 or 3 and sometimes 4 subfractions. In haemorrhagic CNS diseases, Kjellin (1972, 1973a) even found up to 5-6 protein zones in the albumin and prealbumin regions containing bilirubinoids and methaemalbumin compounds, and up to 10 bands in the haemoglobin region with haemoglobin and methaemoglobin compounds. The different patterns of gamma-globulins (a-e, Table 4) found in MS patients seem

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to indicate different rates of production or elimination of certain immunoglobulins Different rates of production might be caused by different viral agents and/or autoimmune reactions. Another explanation might be selective barrier defects for different immunoglobulins; this, however, seems less probable. The former explanation is also supported by the special studies of subfractions within the regions, numbered in accordance with Table 3, as shown in Fig. 5. The "B"-component found by quantitative paper electrophoresis in some cases with "a"- or "d"-patterns on isoelectric focusing is probably caused by more selective barrier damage for smaller protein molecules like albumins, as the barrier damage indicated by quantitative paper electrophoresis is calculated from the albumin concentrations in CSF and serum. In our first series of patients with myelopathy of unknown aetiology (7 cases)isoelectric focusing CSF patterns similar to those found in MS were observed in most cases. This finding is in agreement with the findings of G~.rde and Kjellin (1971) who reported that more than 60~/o of cases of undiagnosed myelopathy may be suffering from MS ; the diagnosis was verified clinically at follow-up examination in about 40 ~,,, of 45 cases examined. In the second series (9 cases) of myelopathy of unknown cause where the isoelectric focusing patterns in the gamma-globulin region were classified as " a " - "f"-patterns (Table 5)~ 7 patients showed similar isoelectric focusing patterns to those found in MS patients but 2 had +'f"-patterns. In the latter 2 subjects the quantitative paper electrophoresis showed a "B"-pattern and increased CSF betasglobulin concentration, respectively, indicating that causes other than MS (barrierdamage and degenerative disorders) exist in a minority of cases of undiagnosed myelopathy (see Ggtrde and Kjellin 1971). The protein fraction found by isoelectric focusing at about pH 7.5 in 3 cases of B ~zneuropathy, disappearing in 1 case examined after treatment of the B t z-deficiency, has not hitherto been studied immunologically. The "I"-pattern found by quantitative paper electrophoresis in 1 of these cases and suspected in another and the "I '+- or "IB"-patterns found by Ggtrde and KjeUin (1971) in 3 cases with myelopathy due to B 1z-deficiency, must be mentioned here, as well as the recent reports on the presence of antibodies in the serum of patients with Btz-deficiency (see G~rde and Kjellin 1971, for refs.). However, the fraction found on isoelectric focusing of the CSF in B~zneuropathy is probably not pathognomonic as we have also observed it in 1 case of encephalopathy, and in 1 case of an optic nerve lesion, both of unknown origin. Unfortunately no B1 z-examinations were performed in these 2 cases. The barrier-damage patterns mostly found by isoelectric focusing of the CSF in cases with polyneuropathy was somewhat remarkable in a single case with GuillainBarr6 polyradiculitis, where the quantitative paper electrophoresis showed a slightly pronounced "BI"-pattern. However, it seems probable that a pathological immunoglobulin pattern would have been found if the CSF had been re-examined in a later phase of the disease. The 6 cases of chronic meningoencephalitis and 1 case of status epilepticus following meningoencephalitis showed isoelectric focusing CSF patterns similar to those of MS or in a minority (2 cases), barrier-damage patterns. However, in some of the cases a study of subfractions in the acid range as well as the immunogtobulin region, showed ~ifferences in the patterns from those found in the MS patients; this suggests the

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possibility of distinguishing cases of meningoencephalitis with an aetiology other than MS. Comparative investigations on the isoelectric focusing CSF patterns in CNS diseases with known virus infections must obviously be of great value. In the few cases of intracranial tumour which were examined, it must be specially pointed out that a "BI"-pattern was found by quantitative paper electrophoresis in a case of acoustic neuroma, while the isoelectric focusing examination showed a clear "f"-pattern. This finding reveals the difficulty in confirming 'T'-components by quantitative paper electrophoresis in cases with high protein concentrations in the CSF. In 1 case of meningioma only a slightly pronounced "f"-pattern was observed by isoelectric focusing, while the quantitative paper electrophoresis showed increased concentrations of prealbumins and betat-globulin in the CSF. However, it seems probable that special studies with a better separation of proteins in the acid region by isoelectric focusing would provide further possibilities for studying the CSF proteins in patients with CNS tumours. Such studies are in progress. Most cases of encephalopathy and hemiplegia of unknown aetiology showed barrier-damage patterns, indicating that MS was probably not the cause of the clinical picture in these cases. The isoelectric focusing patterns of CSF from 3 cases of amyotrophic lateral sclerosis gave no support to the current hypothesis of a slow viral infection being responsible for this disorder. In 2 of the 3 cases barrier-damage patterns were observed. In the 4 cases of cerebellar syndromes of unknown cause, a slight degenerative pattern was observed in 1 case by quantitative paper electrophoresis. In such a case better separation of the acid region on isoelectric focusing might have yielded further information. All cases of cranial nerve lesions showed either "f"-patterns or no significant abnormality, except for 2 cases with optic neuritis and a case with recurrent facial paralysis which showed "d"-pattern~ suggesting a demyelinating aetiology similar to MS. Furthermore, a degenerative pattern was found by quantitative paper electrophoresis in 1 case with tobacco amblyopia. Three cases of systemic disease showed only moderate or slight barrier-damage patterns. In conclusion, isoelectric focusing has been found to be a very valuable method for separating CSF proteins. The thin layer technique in polyacrylamide gel is very suitable for clinical examinations. Possibilities for future development include better separation techniques in acid and alkaline gels, improved densitometric procedures, and finally combinations with other methods such as immunological techniques. Increasing the spectrum of the material from patients with different neurological diseases should improve the diagnostic significance of the different isoelectric focusing CSFpatterns hitherto observed. SUMMARY

The technique of isoelectric focusing in flat beds of polyacrylamide gel which has been found to possess high resolving power for analytical studies of protein mixtures, has been applied to CSF proteins. Besides control subjects with normal CSF-protein

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findings, the clinical material studied predominantly included cases with verified ,~t suspected MS, myelopathies, encephalomyelomeningitis and polyneuropathies. The results of isoelectric focusing of proteins have been compared with those of quantitative paper electrophoresis. The protein fractions obtained by isoelectric focusing were divided into 10 regions. Increased alkaline CSF protein fractions (pH ~8) were found in all cases with MS. The different patterns of gamma-globulins found in MS patients might indicate the different production of immunoglobulins due to different viral agents and/or autoimmune reactions. Differences in the isoelectric focusing patterns of some cases with chronic meningoencephalitis from those found in MS suggests the possibility of using the technique to distinguish meningo(myelo-)encephalitis with an aetiology other than MS. Most cases ofmyelopathy of unknown aetiology showed isoelectric focusing CSF patterns similar to those found in MS. In 3 cases of Blz-neuropathy a fraction at about pH 7.5 was found by isoelectric focusing disappearing in 1 case examined after treatment. ADDENDUM

After this paper was written we became aware of a short paper on isoelectric focusing of CSF proteins [A. L. Latner (1973), Some clinical biochemical aspects of isoelectric focusing, Ann. N.Y. Acad. Sci., 209: 281-298]. The author mentions that "in an unusually high proportion of patients suffering from multiple sclerosis", an abnormal protein spot was found with an isoelectric point slightly more acid than transferrin which travelled somewhat faster than the latter protein during the electrophoresis stage, the spot being also found in other conditions in which demyelination could be expected. This finding could be identical with the double fractions in "region 5" observed by us. The author also found that the IgG zone in CSF from patients with MS "often tended to show a high degree of microheterogeneity'. Normal CSF patterns were obtained in Alzheimer's disease, Huntington's chorea, and idiopathic epilepsy, while the tau protein spot was absent in 2 cases of meningitis. REFERENCES

CLAUSEN,J., T. FOG AND E. R. EINSTEIN (1969) The clinical value of assaying proteins in the cerebrospinal fluid. A comparative study in methods, Acta neurol, scand., 45:513-528. DAVIES, H. (1974) Thin layer gel electrofocusing. In: J. P. ARBUTHNOTT (Ed.), Isoelectric Focusing, Butterworths, London, In press. DELMOTTE, P. (1971) Gel isoelectric focusing of cerebrospinal fluid proteins: A potential diagnostic tool, Z. klin. Chem. klin. Biochem., 9 : 334-336. DELMOTTE,P. (1972) Comparative results of agar electrophoresis and isoelectric focusing examination of the gammaglobulins of the cerebrospinal fluid, Acta neurol, belg., 72: 226-234. DRAWERT, F. AND A. GSRG (1972) Comparative measurements between disc electrophoresis and isoelectric focusing of vegetable proteins in polyacrylamide gels, Chromatographia, 5 : 268-274. FOSSARD, C., G. DALE AND A. L. LATNER (1970) Separation of the proteins of cerebrospinal fluid using gel electrofocusing followed by electrophoresis, J. clin. Path., 23 : 586-589. G~RDE, A. AND K. G. IOELLIN (1971) Diagnostic significance of cerebrospinal-fluid examinations in myelopathy, Acta neurol, scand., 47: 555-568. KJELLIN, K. G. (1969) Kemiska likvorunders6kningar inom neurologin, Svenska Ldk.-Tidn., 66: 3939-3948. KJELLIN,K. G. (1972) Abstract in Proceedings of the Annual General Meeting of the Swedish Society of Medical Sciences, Stockholm, 1972, p. 300.

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