Cerebrospinal fluid enzymology: Creatine kinase, lactate dehydrogenase activity and isozyme pattern as a brain damage index

405

Clinica

Chimica

Acta,

0 Elsevier/North-Holland

89 (1978)

405-409 Biomedical Press

CCA 9692

CEREBROSPINAL FLUID ENZYMOLOGY: LACTATE DEHYDROGENASE ACTIVITY AS A BRAIN DAMAGE INDEX

J.L. VIALLARD

a**, J. GAULME

b, B. DALENS

a Laboratoire 63 003

de Biochimie et d%nzymologie Clermont-Ferrand Chdex (France)

(Received

CREATINE KINASE, AND ISOZYME PATTERN

b and B. DASTUGUE

and b Service

de Pe’diatrie,

a C.H.U.,

April 13th, 1978)

Summary Blood serum and cerebrospinal fluid from a 4.5-year-old girl suffering from convulsive episodes of toxic origin were investigated for lactate dehydrogenase (LDH) and creatine kinase (CK) activities. Elevated levels of both enzymes were found. Furthermore CK was higher in the CSF (680 I.U./l) than in blood serum (160 I.U./l). The CSF activity was demonstrated mainly as the BB form (96%) for CK and H4 (63%) was the predominant form for LDH. Identical investigations were performed 45 h later and results compared with the first set. These data provide an additional example of interest in CSF enzymatic studies as a brain damage index.

Introduction Some enzymes display an isozymic profile relatively specific for cerebral tissue and these could be used as a brain damage index. Elevated lactate dehydrogenase (LDH, EC 1.1.1.27) activity was described in cerebrospinal fluid (CSF) of patients suffering from neurological diseases or injuries of the central nervous system (CNS) [l-3]. Data were reported for total creatine kinase activity (CK, EC 2.7.3.2) in CSF [ 4,5] but few studies included a CK isozyme pattern

[WI. We sodes. in the serum

report the case of a 4.5-year-old girl suffering from toxic convulsive epiAn extremely high CK content composed only of BB dimers was found CSF. LDH activity was elevated and predominantly in the H4 form. The CK and LDH activities were increased but to a lesser extent. The follow-

* To whom correspondence should be addressed.

406

ing day, CSF values decreased but still remained trast serum levels did not vary.

significantly

elevated.

In con-

Methods Enzymatic assays were performed on the supernatant from CSF obtained by centrifugation at 800 X g for 15 min at 4°C and on serum from peripheral blood. Samples were stored at 4°C and assayed no later than 12 h after collection. LDH activity was determined according to the method of Wroblewski [lo] and aHBD (2-hydroxybutyrate dehydrogenase) activity according to that of Elliott and Wilkinson [ll]. Normal ranges were: CSF-LDH 11.5 c 8.6 I.U./l (n = 121); CGFaHBD 9.1 ? 5.8 I.U./l (n = 58); serum-LDH N < 240 I.U./l; serum-cYHBD N < 144 I.U./l. The LDH isoenzymes were analysed by electrophoresis using Cellogel@ (Chemetron, Milano, Italy) followed by a specific staining (Isozyme I LDH from Dade, Miami, U.S.A.). The relative composition was obtained by densitometry using a Chromoscan (Joyce Loebl, Gateshead, England). The total CK activity was determined at 25°C using Statzyme@ CPK n-l (Worthington, Freehold, U.S.A.) based on the method of Rosalki [12]. Normal values were: serum <50 I.U./l, CSF
407

TABLE

I

ENZYMATIC

DETERMINATIONS

Cerebrospinal Normal No.

1

No.

2

LDH

Total

(1.U.P)

(1.U.P)

CK

(%

fluid 11.5

f 8.6

315 101


100

680

91

19.5 -97%

<240

<50

0

No.

1

1476

160

13.5

No.

2

1440

115

18.5 -

Variation

-1%

* See ref.

TABLE

*

Residual values

CK)

97.5

-6%

Serum Normal

*

of total

Activity

values

Variation

CK-BB

-28%

activity

(o/o)

8.

II

CEREBROSPINAL Cerebrospinal

FLUID: fluid

LDH

ISOENZYMES

Composition

(%)

H4

H3M

%Mz

No. 1

63

28

6

2

1

No.

56

32

6

4

2

2

TABLE

HM3

M4

III

CEREBROSPINAL

FLUID:

RESULTS

OF

CYTOBIOCHEMICAL

DETERMINATIONS

Cerebrospinal

Appear-

Prokin

Glucose

Chloride

Blood

Neutro-

Red

fluid

aIUX!

(lzfl)

(~~wu

mmom

white

phik

(N/1)

cells

(%)

cells

(N/I) No.

1

Clear

0.96

13.8

135

No.

2

Clear

0.32

-

-

95x106 8X106

95 -

1 x 106 375

x 106

DEAE-Sephadex chromatography, 96% of the recovered activity was estimated as due to BB component. Residual activity was 97% after using anti-M antisera. Increased LDH (315 I.U./l) and (rHBD (225 I.U./l) activities were also found in CSF. Isozymic pattern showed a clear predominance of the H4 form representing 63% of the total activity (Table II). The cytobiochemical examinations showed a slightly increased protein content and some neutrophils (Table III). Two days after admission, the child was comatose. Serum CK activity exhibited a slight decrease. The activity attributed to B-containing subunits was 18.5%. The LDH and aHBD values were identical to the previous determinations. In the CSF the CK showed a striking decrease, to only 3% of the previous assay. Use of anti-M antisera demonstrated 97.5% CK activity as BB dimers. The LDH (107 I.U./l) and aHBD (79 I.U./l) values decreased much less. The

408

isozymic pattern showed the prevalence of Hq. The protein content of the CSF was within the normal range. Some erythrocytes were present but the supernatant was not x~thochromic, Death occurred 72 h after admission with postmortem examination showing no macroscopic abnormality (no haematoma, no bleeding). Discussion There were several ch~acteristics of CK activity observed in the CSF during this case. The enzyme activity was very high. Few observations have revealed such elevated values [6,9]. Nearly all of this activity was attributable to the BB form, characterising its brain origin. In contrast, serum values were lower than the CSF displaying an isozymic pattern in which MM dimer was the chief component. This tends to refute the possibility that CSF values increased following diffusion from the serum with an increased permeability of blood-CSF barrier. Furthermore, the increased CSF activity occurred during the convulsive episode and close to the toxic attack. The fall in enzymatic activity was very acute to 3% of the initial value 24 h later. We could not detect in the second CSF specimen the presence of an inhibitor of CK. This drastic evolution is in agreement with previous observations [8,9] and with experimental data on cats [15f. Such a decrease coufd explain contradictory results when a possible correlation between increment of activity and neurological observation was studied. Although sixteen times higher than the normalvalue the LDH activity increased to a lesser extent than the CK and 44% was still present 24 h later. Isozymic separation for both CSF specimens showed prevalence of the fast components in agreement with LDH cerebral tissue content. Thus the two enzymatic activities studied in the cerebrospinal fluid showed very marked increases and the isozymic patterns signified cerebral origin. In contrast to CSF data, serum LDH and CK elevations could not be characterised as originating from CNS tissue. Furthermore the absence of variation for LDH and the lower decrease for CK activity indicated poor correlation with the CNS process. In conclusion, the present data are an especially good example of the great relevance of enzymatic studies during acute neurological syndromes as compared to usual CSF biological examinations. Furthermore they outline the very large range of variation and rapid changes of CK activity in this fluid. The latter justifies the concomitant use of parameters such as LDH, perhaps less specific but showing slower rates of variation, to define more clearly brain damage through CSF enzymatic profiles. References 1 2 3 4 5 6

Be&y, H.N. and Oppenheimer, S. (1968) New En& J. Med. 279.1197-1202 Nelson, P.V., Carey. W.F. and Pollard. A.C. (1975) J. Cli. Pathol. 28,828~833 Neches, W. and Platt. M. (1978) Pediatrics 41,1097-1103 Katz, R.M. and Liebman, W. (1970) Am. J. Dis. Child. 120.543-546 Nordby, H.K., Tveit. B. and Ruud, I. (1975) Acta Neurochir. 32.209-217 Vaycr, P.M., Gabl, F.. Granditsch, G., Widhalm. K., Zyman, H. and Dcutsch, E. (1976) 22.1405-1407

Cliu. Chem.

409 7 Banerji, A.P., Khopkar. P.P.. Deshpande. D.H. and Desai. A.O. (1973) CIin. Chii. Acta 43.431-434 8 Sherwin, A.L., Norris, J.W. and Bulcke. J.A. (1969) Neurology 19.993-999 9 Wolintz, AH., Jacobs, L.D.. Christoff. N.. Salomon. M. and Chernick. N. (1969) Arch. Neural. 20, 5441 Wroblewski, F. and La Due, J.S. (1955) Proc. Sot. EXP. Biol. 90, 210-213 Elliot, B.A. and Wilkinson, J.H. (1961) Lancet i. 698-699 Rosalki. S.B. (1967) J. Lab. Clin. Med. 69. 696-705 Mercer, D.W. (1974) CIin. Chem. 20, 36-40 Neumeier, D., PreIlwitz, W.. Wurzburg, V.. Prundobler, M.. Olbermami, M.. Just, H.J., Del, M. and Lang, H. (1976) Clin. Chim. Acta 73.445-451 15 Maas. A.I.R. (1977) J. Neurol. Neurosurg. Psych&r. 40.655-665

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