D. Karcher

THE DIFFERENTIAL DIAGNOSIS OF NEUROLOGICAL DISEASES BY FRACTIONATING ELECTRO-PHORETICALLY THE CSF γ-GLOBULINS*

ELECTROPHORETIC studies have shown qualitative and quantitative changes in the proteins of the cerebrospinal fluid in certain neurological conditions. Since the yglobulins are almost always affected, the present report deals primarily with this fraction. A specific abnormal distribution of the CSF y-globulins has been reported by BOOIJ (1 958) in cases of subacute sclerosing leucoencephalitis (SSLE). LOWENTHAL (1959) has also pointed out that in the common infectious types of encephalitis, some of the y-globulin fractions migrate at a slower rate than in the normal CSF. It therefore appeared that, besides being usually increased in quantity, the y-globulins might also show qualitative changes in diseases such as SSLE, multiple sclerosis, syphilis and African trypanosomiasis. The electrophoretic pattern on agar gel of the CSF proteins in cases of multiple sclerosis differs from the normal in that there is an increase in the y-globulin fraction, which is most striking in the region of slow migration. In SSLE, two y-globulin subfractions migrate further towards the anode than in the normal, while in African trypanosomiasis the increase of the y-globulins is more pronounced and more diffuse than in either multiple sclerosis or neurosyphilis, and without the presence of the slow fractions found in SSLE (Fig. 1). These findings suggested that differentiation between multiple sclerosis, SSLE, neurosyphilis and African trypanosomiasis might be made on the basis of the electrophoretic pattern of the CSF proteins. Two methods were used to establish these different patterns: (1) The determination of the rate of relative mobility (m,) of the various fractions. (2) The determination of the relative concentrations of each of the fractions by densitometry.

MICRO‐ELECTROPHORESIS IN AGAR GEL OF PROTEINS OF THE CEREBROSPINAL FLUID AND CENTRAL NERVOUS SYSTEM

IN previous studies of the protein distribution in neurological dise%ses by paper electrophoresis, we found an increase in various conditions in the y-globulins of the cerebrospinal fluid (CSF). The chemical investigation of the pathological y-globulins in these conditions, showed that they do not always have the same chemical composition. Thus we were able to show that in the CSF of some encephalitics the y-globulins contained a high proportion of glycoproteins (JANSSENS et ul., 1958). In view of the importance of the y-globulins in syndromes such as multiple sclerosis, neurosyphilis and subacute sclerosing leucoencephalitis, we sought a method that could differentiate between the different y-globulins. Electrophoresis in agar gel was successful in separating the y-globulins of the CSF proteins with far better results than on paper. Paper electrophoresis was carried out by the procedure of GRASSMAN and HANNIG (1950). The protein samples applied at one end of a horizontal strip migrated towards the anode. We found that although the serum proteins applied in the centre of the agar gel migrated partially towards the anode and partially towards the cathode, the results were quantitatively similar with both methods. The same technique was also applied to the proteins of the central nervous system.

Heterogeneity of lactic and malic dehydrogenase in cerebrospinal fluid.

RESEARCH on enzymic activity in the cerebrospinal fluid (CSF)t has shown that there is an increase in glutamic-oxalacetic transaminase activity in cerebral arteriosclerosis and in subacute sclerosing leucoencephalitis; in some lipidoses the aldolase (ALD) activity is increased (ARONSON, SAIFER, PERLE and VOLK, 1958). Compared to the serum, the ratio of enzymic activity to total protein concentration is greater for the CSF. Although the lactic dehydrogenase (LDH) and ALD activity of the CSF is about one tenth of that of the serum, the total protein of the CSF is about 1/200 of that of the serum. The amount of LDH varies with the CSF cell count and not with the total protein concentration. Recent investigations on enzymes have introduced the concept of tissue specificity. As a corollary of this, a heterogeneity of serum enzymes has been shown by chemical, chromatographic, and especially by electrophoretic techniques. WIEME’S (1959) method called ‘enzymoelectrophoresis’, makes it possible to demonstrate the heterogeneity of LDH and malic dehydrogenase (MDH) in human serum. This method was applied in a preliminary study of the heterogeneity of these enzymes in the CSF (LOWENTHAL, VAN SANDE and KARCHER, 1960) and more detailed results are now given. REAGENTS A N D M E T H O D

Serum gamma globulins in 84 typical cases of subacute sclerosing panencephalitis.

Iri 1962, we first reported that there was evidence of well-pronounced M components in the gamma globulin field, separated on agar gel by electrophoresis, in the sera of patients affected with subacute sclerosing panencephalitis (SSPE).l This was confirmed in our laboratory in 19642 and 1968.3 It has also been confirmed in many other studies concerned with this neuroimmunopathologic di~order.~-g Cellulose acetate electrophoresis revealed similar gamma globulin patterns. Our results were later confirmed by KolarlO and Peter and Lowenthal.11 Our attention has again been drawn to this subject by two recent papers concerned with the serum gamma globulins in SSPE: 113 Jabbour et described an additional slow cathodic band in the serum gamma globulins separated on cellulose acetate by electrophoresis and [ 2 ] Freeman13 stated that the serum gamma globulins of SSPE subjects are in normal relative concentrations.

ELECTROPHORETIC PATTERNS OF LACTATE DEHYDROGENASE ISOENZYMES IN NERVOUS TISSUES

IN PREVIOUS publications (LOWENTHAL, 1961, 1962; LOWENTHAL, VAN SANDE and KARCHER, 1961 a and b, 1962; LOWENTHAL, VAN SANDE, KARCHER and RICHARD, 1962) it was shown that the distribution of the multiple molecular forms of lactate dehydrogenase (LHD) varies in different parts of the brain. The present experiments were made in an attempt to clarify the LDH variations in different parts of the human nervous system and to find out if there was a relation between the distribution of LDH isoenzymes and the different fractions of the water-soluble proteins. The main investigation is of the soluble protein extracts from spinal cord and peripheral nerve.

Electrophoretic studies of central nervous system proteins

Abstract Paper electrophoresis of cerebrospinal 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 electrophoresis. These preliminary results led us then to attempt to study more specifically the qualitative differences. Agar electrophoresis reveals a more detailed subdivision of cerebrospinal 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 origin of those protein fractions. Additional studies comparing agar electrophoretic patterns obtained from human and from animal cerebral tissues reveals an essentially similar pattern.

Protéinogrammes et enzymogrammes des protéines musculaires en pathologie humaine

Abstract By electrophoresis in agar a study was made of the water-soluble proteins and lactate dehydrogenase of the muscular tissue in 33 fragments of human muscles obtained by biopsy or autopsy. Normal subjects, myopathies, cases of Steinerts disease and of neurogenic amyotrophia were studied and the results compared. The proteinograms make it possible to confirm that alterations are observed in albumen and m globulins which are characteristic of the muscular tissue. The alterations are especially marked in Steinerts disease, they are relatively less marked in neurogenic amyotrophia. The fastest globulin fraction seems to be also the most resistant to the alterations observed. In the enzymograms of lactate dehydrogenase, the alterations observed in myogenic and neurogenic amyotrophias are alike. In Steinerts disease the results obtained are practically normal.

DEVELOPMENTAL PROCESSES AND PROTEINS AND LACTATE DEHYDROGENASE ISOENZYMES IN THE HUMAN BRAIN

The question has often been raised as to the criteria defining maturation of the central nervous system.

Protein Fractions and Lactico-dehydrogenase Isozyme Distribution in Normal and Pathological Nervous Tissue (Man and Animal)

Publisher Summary This chapter describes the application of the electrophoresis method to the study of the central nervous system in normal and pathological conditions in man and animal. Electrophoresis in agar gel, in particular, was found to be an adequate technique. Besides the great resolving power of this medium, the micro-scale application of this technique to the cerebrospinal fluid (CSF) makes it suitable for routine assay in laboratory work. The method was found to be applicable to the study of the hydrosoluble proteins of the central nervous system and to reveal the multiple molecular forms of various enzymes. The chapter provides results obtained from the study of the central nervous system. The study of the enzyme structure shows that in demyelinating diseases the total malic dehydro genase (MDH) and lactico-dehydrogenase (LDH) activity is increased in white matter. The fractionation of LDH yields five fractions with relative mobilities that are species-specific. The quantitative distribution of these multiple molecular forms for a same species is of four different types. Pathological material reveals no changes either in the relative mobilities or in the quantitative values expressed in per cent. The results differ from those found in other tissues. Indeed the protein and isozyme pattern seems to be tissue-specific and until now no other types have been discussed. These findings show the complexity of the central nervous system and stimulate to a further topographical investigation.