albumin index

195

Clinica

Chimica

Acta,

105

@ Elsevier/North-Holland

(1980) 195-199 Biomedical Press

CCA 1423

CSF TRANSFERRINS CHARACTERIZED TRANSFERRIN/ALBUMIN INDEX

L. KERfiNYI,

MARIA

KOLTAI

Department of Neurology Rit u. 2. (Hungary)

(Received

June 18th,

BY THE

and I. SZIRMAI

and Psychiatry,

University

of P&s,

Medical

School,

H-7623

PCcs,

1979)

Summary Unconcentrated CSF and 200 times diluted serum samples from 192 patients were examined by agar electrophoresis; their albumin and transferrin content were determined simultaneously by radial immunodiffusion. Using the data on transferrin and albumin concentrations in serum and cerebrospinal fluid, the transferrin/albumin index was calculated in a similar way to the IgG/albumin index. Normally the transferrin/albumin index is 1.68 (n = 77; S.D. = 0.22). If the permeability of the blood-CSF barrier increases, the transferrin/albumin index gradually decreases to 1. If the tau-globulin fraction is increased on the agar pherogram, the transferrin/albumin index will be greater than 2.34 @ + 3 S.D.).

Introduction The beta-globulins of the cerebrospinal fluid (CSF) can be separated into beta,, beta2 and tau-globulin fractions by agar electrophoresis [ 11. One of the transferrin (TR) variants originating from the serum migrates in the beta, fraction. The bulk of the tau fraction consists of another TR variant which is not detectable in serum. After neuraminidase treatment beta,-TR moves in the tau position. Therefore, the two TR variants differ only in the content of neuraminic acid [ 21. Immunologically, both TRs are identical, which explains why the TR precipitate has two peaks on the immunoelectrophoretogram [3]. The origin of the tau fraction has not yet been clarified, which might chiefly be due to methodological difficulties. In the present paper a simple method based on radial immunodiffusion (RID) will be proposed for studying CSF TRs.

Correspondence should be addressed to Dr. L. KerBnyi. Department sity of PBcs. Medical School, H-7623 P&s. R&t. u. 2,, Hungary.

of Neurology

and Psychiatry,

Univer-

196

Materials

and methods

From the routine agar pherograms of about 600 CSF samples, 192 were selected irrespective of diagnosis. The samples had to fulfil the following criteria: (1) CSF should be free from red blood cells and traces of hemoglobin (examined by Sangur-Test of Boehringer Mannheim GmbH, F.R.G.); (2) the band of the tau-globulin fractions should be unambiguously recognisable on the pherograms, irrespective of whether they were increased, decreased or normal. Agar electrophoresis was performed without concentration of the CSF samples in a home-made perspex chamber cooled to 4°C with petroleum ether [ 41, and the pherograms were stained with silver [5,6]. The total protein content of the CSF was measured by the method of Rieder [ 71. The TR and albumin (ALB) content of the serum and unconcentrated CSF of the 192 selected patients were determined by RID [8]. The CSF samples were obtained by lumbar puncture, except in 5 samples which were taken suboccipitally. From the concentration of ALB and TR in the serum and CSF, the TR/ALB index was calculated using the following formula: TR/ALB

CSF TR/serum -~ CSF ALB/serum

index:

Experimental

TR ALB

=.CSFTnX_ serum TR

serum ALB _ CSF ALB

results

The tau-globulin fractions on the agar pherograms were normal or decreased (“mixed-type”) in 149 of the total of 192 samples. This group was further divided on the basis of the total protein content of the CSF (see Table I). The tau-globulin fractions were increased in the agar electrophoretograms in 38 cases. Table II shows the values for the TR/ALB indexes of this group. The clinical diagnoses of the above 38 cases were added to the collection after their selection. The TR/ALB indexes of the 5 suboccipital samples are also included in Table II.

TABLE

I

TR/ALB

INDEXES

IN THE Agar Total

CSF

OF

electrophoresis protein

Number TRIALB Mean

BY

in CSF

in CSF.

of patients

mg/l (n)

WITH

AGAR

NORMAL

OR

DECREASED

TAU-GLOBULIN

ELECTROPHORESIS

norma

mixed-type

<500

501-1000

1001-2000

>2000

77

43

21

8 **

index (ZJ

1.68

Standard

deviation

Standard

error

* Upper

PATIENTS

OBTAINED

limit

* * Individual

(sx)

(5)

of normal: values:

1.23

1.14

1.04

0.22

0.19

0.15

0.07

0.03

0.03

0.03

0.02

X + 3s,

*

= 2.34.

1.13-1.07-0.96-1.10-0.96-1.00-1.00-1.09.

FRACTIONS

197 TABLE

II

TR/ALB

INDEXES

OBTAINED ~-__ Clinical

BY

OF AGAR

PATIENTS

WITH

ELECTROPHORESIS

INCREASED (TOTAL

diagnosis

Number

TAU-GLOBULIN PROTEIN

OF

TR/ALB

FRACTIONS CSF

SAMPLES:

IN 100-240

THE

CSF mg/l).

indexes

of cases -Chronic

alcoholism

12

2.50-2.54-2.60-2.96-3.02-3.12-3.39-3.404.55-4.58-5.40-9.18

Symptomatic

epilepsy

6

2.51-2.66-2.67-2.84-3.14-4.27

Encephalopathy

5

2.59-3.00-3.46-3.49-3.70

Cerebral

atrophy

5

2.35-2.41-2.60-2.80-3.13

Multiple

sclerosis

4

2.39-2.88-4.40-8.84

Retrobulbar

neuritis

Progressive

multifocal

Funicular

ieukoencephal~pathy

myelosis

Taboparesis Suboccipital

liquors

(normal)

3

2.88-3.44-3.60

I

6.47

1

3.70

1

3.64

5

2.35-2.45-2.56-2.81-2.96

Discussion The pathological changes in the agar pherograms were estimated by two independent examiners by the naked eye. As compared with the beta, fraction, silvering of the tau-globulin fraction in normal individu~s was found to be less intense (see Fig. la). If the tau-globulin fraction is more intensely stained than the beta,-globulin fraction, the pherogram is considered to be of the tautype (see Fig. Ib). It should be pointed out, that the increase in mu-globulin does not always seem to be so clear-cut, as in Fig. 1. Therefore, an attempt was made to express it numerically in accordance with the IgG/ALB index first used by Link and Tibbling [9]. It was assumed, that the elevation of tau-globulin fraction in the agar pherograms is, in fact, indicative of an increase in autochtonous CSF TR. However, it is only the total of the two TR variants, that is determined by RID. That is why the TR concentrations in the CSF and serum were related to the serum and CSF ALB concentrations in exactly the same manner as in the case of the IgG/ALB index. Theoretically, the TR/ALB index should be greater than I in accordance with the well-known fact that some of the CSF TR originates from the central nervous system [ 101. This was actually the case in 77 patients

a

b Fig.

1. Normal

(a) and

tau-type

(b)

agar pherogmm.

198

in whom CSF agar electrophoresis and total protein were normal; the mean value of the TR/ALB index reached as much as 1.68 (Table I). This value represents the total amount of TR coming from the blood and released intracerebrally under normal conditions. The distribution of the 77 cases is seen in Fig. 2. Except for the two peaks around 1.95 and 2.15 the distribution may correspond either to the Gaussian or to the log-normal type. This question was not investigated in detail, but it follows from the diagram that patients with normal CSF electrophoresis and normal total CSF protein have TR/ALB indexes lower than 2.34 @ + 3 S.D.). If the blood-CSF barrier is damaged, i.e. total CSF protein increases and the tau-globulin fraction decreases in the agar pherogram (“mixed type”), the TR/ ALB index will also decrease progressively (Table I). In borderline cases the TR/ALB index = 1, the tau-fraction is not visible in the agar pherogram, therefore the proportion of TR to ALB will be the same in serum and CSF. In the n = 8 ** group (see Table I) the individual values are scattered around 1, this also indicates that the mean value is correct, in spite of the small number of cases. In the cases of barrier impairment this finding has no clinical significance, because the measurement of total protein and TR/ALB index have the same meaning. From a pathophysiological point of view, however, the decrease of TR/ALB index, supporting the fact that autochtonous TR falls as total protein rises in CSF, is important (the accruing investigations are in preparation). In patients with increased tau-globulin fraction in the CSF agar pherogram, the TR/ALB index exceeds 2.34 (Z + 3 S.D.). These data and the clinical diagnoses collected subsequently are given in Table II, which contains; as a comparison, the TR/ALB indexes determined in the 5 suboccipital liquors too. In all suboccipital samples the TR/ALB indexes were higher than 2.34 but lower than 3.00, which corresponds to the fact that suboccipital CSF contains more tau-globulins than lumbar CSF [ll]. Detailed clinical analysis of the cases included in Table II and collection of similar ones are in progress. It seems to be characteristic that the protein content in the CSF samples is generally less

Fig.

2. The

distribution

of TR/ALB

indexes

in the

control

group

(n = 77).

199

than 200 mg/l and the ALB content does not exceed 100 mg/l. It should be pointed oet, however, that the low ALB concentration itself does not cause an increased TR/ALB index, as it was found in many CSF samples containing ALB in lower concentration (unpublished data). The data in the literature concerning increase or decrease of the tau-globulin fraction in pathological conditions are contradictory; Lowenthal [ 121 suggests therefore further investigation into the pathophysiology of the tau-globulin fraction. In our opinion the contradiction is caused in part by the fact that the beta, and the tau fraction are not always separated by the examiners. All our agar pherograms obtained by simultaneous examination of several thousands of serum and CSF specimens contain regularly beta1 and beta2 fractions as well as betal, beta2 and tau fractions in the serum and CSF respectively, except the mixed-type ones [ 131. Unlike Verheecke [ 141 we have found the electrophoretic mobilit,y of t,he tau fract,ion t,o he constant. Tn a few cases (13 from a total of 2300) a simultaneous duplication of the beta1 fraction as well as of the beta, and the tau fraction in the pherograms of the serum and CSF from the same patient has been observed [ 131. (The double beta, fraction, i.e. double TR of the serum is mentioned as a genetic variant by Johansson [15].) Some pherograms from cases of cerebral atrophy contain two or three intermediate bands between the beta, and tau fraction; the electrophoretic mobility of the two fractions (i.e. beta, and tau), however, remained constant in these cases too [ 131. As a result we have found the quantitative change in the tau-globulin fraction to correlate well with the change of the TR/ALB index. Five samples have been kept for 3 weeks at +4”C and the following 3 months at -8°C; their pherograms and TR/ALB indexes obtained on re-examination were the same as before being stored. Acknowledgements This research was supported by the Scientific Research Council of the Hungarian Ministry of Health (3-20-0303-03-2-P). The authors are indebted to Mrs. A. Krisztics, Gy. KAlsecz, A. Horv&h and Miss M. Szab6 for expert technical assistance. References 1

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