Acta neurol. scandinav. 57, 358-365,1978 Departmelnts of Clinical Chemistry and Neuro~logy,University of Oulu, and Kivela Hospital, Finland
Measurement of reference values for certain proteins in cerebrospinal fluid A. AHONEN, V. V. MYLLYLA AND E. HIOKKANEN Reference values are presented for certain cerebrospinal fluid proteins and related indexes, based on determinations carried out on 46 patients with diffuse lower back pain using radial immunodiffusion and electroimmunoassay. A sex difference was noted in total protein and albumin values. The electroimmunoassay method with its sensitivity enhanced by the use of Coomassie Blue staining and optimal antiserum concentrations enabled a reference value to be determined for IgA in cerebrospinal fluid. Key words: Cerebrospinal fluid proteins - electroimmunoassay - immunoglobulins - radial immunodiffusion - reference values
Despite its nonspecificity, the measurement of the total protein concentration in CSF is still regarded as useful in clinical practice, although an elevated protein content may be associated with a large number of quite disparate diseases. It has been suggested that this procedure should be replaced by CSF albumin determination, as measured CSF albumin levels vary much less from one laboratory to another than do values for total proteins, due to the more reliable methods and standards for calibration. Many other protein fractions of CSF have also been extensively discussed in the literature, but only one seems at present to have any diagnostic value, namely 7’globulin (principally the IgG portion), when its concentration is high (Fishman 1975). The colloidal reactions in common use earlier as indirect indicators of increased IgG concentration have been replaced by more specific techniques such as electroimmunoassay and radial immunodiff usion, which are also simpler for practical use than gel or filter paper electrophoresis. The purpose of the present work was to establish reference values for CSF total proteins, albumin and immunoglobulins and for certain indicative ratios, and to compare the results of radial immunodiffusion (RID) with those of electroimmunoassay (EIA).
359 PATIENTS AND METHODS The investigation was carried out on 27 men and 19 women at a clinic belonging to the Orthopedic Hospital of Invalid Foundation in Helsinki, Finland. They varied in age from 19 to 55 years with a mean of 39 years (mean 39 and median 41 for men, mean 38 and median 39 for women), and all had a history o t lower back pain for which they had been treated with various analgetic and antiinflammatory drugs and for which a lumbar myelography had been carried out. They included all the patients with negative findings in myelography or in operative (discus) exploration. There was no evidence or subjective complaint of any other neurological disease. Nor was evidence of lateral discus herniation obtained during the later follow-up time of 1 year. A routine lumbar puncture was carried out in a sitting or supine position and 2-3 ml of CSF was removed before injection of the contrast medium. A serum sample for similar laboratory tests was taken at the same time in 30 cases. The samples were stored immediately at -2OOC (CSF without centrifugation) and analyzed at the Kivela Hospital, Helsinki, Finland, within 2 to 6 weeks of sampling, during which period no significant alterations in protein levels were observed. Total protein concentration was measured by the routine biuret method, in which a c o b r complex binding between alkaline copper salt and protein is measured colorimetrically. Albumin and immunoglobulins were first determined by the standard RID method (Mancini et al. 1956, Fahey & McKeZvey 1965), using commercially available (Behringwerke, Marburg Germany) low concentration immunodiffusion agar plates for CSF analysis (PartigenQ. The second method involved immunoelectrophwesis (Sclzeidegger 1955, Laurel1 1966) using research grade EIA plates (ExaPhorB, Orion Diagnostica, Helsinki, Finland) further modified to obtain greater sensitivity and suitability for the present CSF investigations. EIA analyses were carried out oa a thin agar gel, which is a buffer substance also containing the antibody. The samples were first treated with ExaPhor formulant (formaldehyde) to change the electrical charges of the immunoglobulins to improve the migration (Teppo 1973) and after 10-20 min were placed in small circular wells punched in the gel layer. An electric field of 5 V/cm was then applied over the gel to incite electrophoretic migration of the antigen. The area of precipitate formed in the antigen-antibody reaction (rocket shape) is thus proportional to the amount of antigen in the sample (Weeke 1968, Laurel2 1972). The time needed for the formation of this precipitate was 3 h for albumin, 5 h for IgG and IgA and 6 h for IgM. The sensitivity of the plates for the detection of low concentrations of antigens in CSF was improved by Coomassie Blue staining and by optimizing the antiserum concentration, which showed to be 0.4-1.2 mg/l. The protein concentrations are expressed as milligrams per liter (mgll). The Student’s t-test and paired t-test were used.
RESULTS
The mean total CSF protein concentration (and SD) for the 46 patients was 363 k 70 mg/l (median 336) (Fig. la) that for the males being 378 k 69 mg/l and that for the females 337 66 m a . An almost significant difference ( P < 0.06) existed between the sexes, but no dependence on age could be derived from the present series. The CSF albumin and immunoglobulin levels are shown in Table 1. The respective albumin values were 187 k 58 mg/l (RID) 198 67 (EIA) and those for IgG 21.0 7.0 mg/l (RID) (Fig.
*
*
*
46 29 17 46 29 17 46 29 17 46 29 17
187 2011 166 21.0 20.6 21.8 5.7% 5.4 6.2 12.0 10.0 13.0
Means
11.3
5.9%
22.0
188
Medians 71-303 90-312 52-279 7-35 7-34 7-37 2.4-8.9 % 2.0-8.8 3.0-9.5 3.2-21.0 2.4-18.5 4.1-22.0
Reference values
Radial immunosdiffusion (RID)
46 29 17 46 29 17 302 13 17 30 13 17
No. of patients
P < 0.05 when compared with the corresponding value for the females. The lower number of patients in this group was due to small amount of CSF sample obtained.
Albumin males females IgG males females IgGItotal protein males females IgGIalbumin males females
No. of patients 198 205 186 25.0 24.3 26.1 5.2% 5.6 5.0 11.0 12.2 10.1
Means
10.8
5.3%
23
180
Medians
64-332 76-334 42-330 16-34 16-32 17-35 2.4-8.0% 3.6-7.6 1.8-8.2 2.3-20.1 7.0-17.3 0.0-2 1.2
Reference values
Electroimmunodiffusion (EIA)
Table 1 . Comparison of CSF albumin and immunoglobulin concentrations ( m g / l ) and various indexes immunodiffusion and electroimmunoassays. Means, medians and reference values (means & 2 S.D.)
0
o\
w
361
12
t .-LA--
36 39 MGlL
Figure 1 . Histograms f o r CSF total proteins ( a ) and IgG ( b ) concentrations of the present material.
l b ) and 25.0 2 4.3 mgA (EIA). The difference between the albumin values obtained by the two methods was not statistically significant, but the IgG concentrations obtained by RID were markedly lower than those given by EIA ( P < 0.01). The albumin values were lower in the females than in the males ( P < 0.05 with RID, P > 0.05 with EIA), but no significant sex differences were observed in the IgG concentrations. The ratios of IgG/total protein and IgG/albumin in CSF are also shown in Table 1. No statistical differences were observed here between the male and female patients. Correspondingly, Table 2 presents the CSF/serum ratios for IgG and albumin concentrations. Unfortunately EIA determinations could be carried out in this case on only eight patients, due to the small amount of sample taken. IgA and IgM concentrations in CSF were also measured in 12 of the patients using the same two methods, but the only detectable concentration was a reference value of 1.5 2 1.3 mg/l for IgA given by EIA.
362 Table 2. Comparison of CSF/serum ratios (promilles) f o r I g G and albumin obtained by radial immunodiffusion and electroirnmunoassay. Means and reference values (means k 2 S.D.)
RID No. of Means patients CSF-IgG/serurn-IgG males females CSF-albuminkerum alb. males females
30 12 18 30 12 18
1.7%0 1.6 1.8 5.0 4.8 5.1
EIA
Reference No. of values patients
Means
0.5-2.9%0 0.4-2.8 0.4-3.2 2.G8.0 2.0-7.6 2.1-8.1
8
1.7%0
0.7-2.7 550
8
3.7
2.0-6.0
Reference values
The reference values calculated from the present material are listed in Tables 1 and 2. Since reference values calculated by Student’s t-test and nonparametric tests did not noticeably differ from each other the former values were used. The skewness and kurtosis of the distribution of the results were statistically tested, but no significant difference from Gaussian distribution was observed. Two examples of histograms are presented in Fig. 1. DISCUSSION
Since for ethical reasons the authors believe that CSF samples should not be taken from healthy subjects, the present work was carried out entirely on patients with diffuse lower back pain in whom no evidence of herniation of an intervertebral disc could be verified in subsequent investigations (myelography or exploration and follow up). As the patients showed no signs of any other neurological disease, they may serve in our opinion as a “normal” reference group, although all minor abnormalities such as slight radiculitis or drug effects can not be excluded. The reference value of 223502 mg/l obtained here for CSF total proteins lies in the same range as those reported by Tourtellotte (1959) and Mertin et al. (1971), who used the biuret method. The almost significant differences found between the sexes by these authors seem to be due principally to differences in albumin levels, as can also be seen in the present material. The CSF total protein concentration was lower in the women than in the men, as also noted by Izikowitz (1941) and Dencker (1961, 1963), for example. On the other hand there are some investigators who have not found any sex difference in CSF protein levels (Rieder et al. 1962, Arnold 1966, Jung et al. 1973, Tibbling et al. 1977). This may be due to differences between patient groups, for it is known that protein levels in CSF are higher in older age groups than in younger ones, probably due to impaired function of the blood-brain barrier
363 (Rieder et al. 1962, Arnold 1966). The present material was not representative in that respect. Albumin is the largest protein fraction in CSF (cf. our reference value of 64-332 mg/l) and is formed outside the nervous system. It has been suggested that the CSF albumin level may give important information on CSF dynamics, with high levels referring to lesions of blood CSF barrier and/or reduced CSF turnover (Delpech & Lichtblau 1973, Ganrot & Laurell 1974). The sex differences appear to be clearer in the albumin values than in CSF total proteins, but the fact that the ratio between CSF and serum albumin levels is in the same range in both sexes suggests a dynamic equilibrium between the CSF and serum proteins. Since more reliable determination techniques are available, CSF albumin might well be preferred to total proteins. In addition, it would enable the CSF/serum ratio for albumin to be used as an index of blood-brain barrier function, in which even alterations in serum albumin levels will be taken into account (Delpech & Lichtblau 1973, Link et al. 1977a). The IgG concentration in CSF obtained here (735 mg/l) lies between the reference ranges reported by Glasner & Wenig (1973) and Ganrot & Laurell (1974) but lies in the same range as those given by Tibbling et al. (1977) and EickhofJ & Heipertz (1977) and shows no clear difference between the sexes. An elevated CSF/serum ratio of IgG is generally accepted as reflecting local IgG synthesis in the central nervous system (Ganrot & Laurell 1974), the usefulness of which seems to be proven in the diagnostics of multiple sclerosis, for instance. If simultaneous serum IgG values are not available, the local IgG synthesis in the central nervous system can be estimated from the ratio of IgG/total proteins or IgG/albumin in CSF (Delpech & Lichtblau 1973, Link et al. 1977b). No detectable concentrations of IgA and IgM could be shown by the RID method, and the EIA method gave a reference value for IgA, but not for IgM. Similar results have been reported by other investigators (Delank 1975, Laterre 1975), although IgM concentrations in CSF have been measured in pathological cases (Greenwood 1973, Whittle et al. 1977). The present results show that both methods are suitable for CSF albumin and IgG determinations. RID is easier to perform in routine work, but when very low concentrations, e.g. of IgA and IgM, are expected we would recommend ETA, which also has the advantage of requiring a shorter time. ACKNOWLEDGEMENTS The authors wish to thank Drs. K. Touru-Kaisila and K . Ojala for their assistance during the investigation. The Partigen plates were kindly donated by Hoechst Fennica, Helsinki, Finland and the ExaPhor electroimmuncrassay plates by Orion, Helsinki, Finland.
3 64 REFERENCES Arnold, F. (1966): Liquor-Untersuchungen an Gesunden und Multiple Sklerose-Kranken. Schweiz. Med. Wschr. 96, 1098-1103. Deland, H. W. (1971): Die Immunoglobuline im Liquor cerebrospinalis und ihr klinischdiagnostische Aussagewert. Nervenarzt 42, 483-490. Delpech, B. & E. Lichtblau (1973): Etude quantitative des immunoglobulines G et de l’albumine du liquide cCphalorachidien. Clin. Chim. 37, 15-23. Dencker, S. J. (1963): Studies on variation of CSF total proteins and cells with sex und age. Kungl. Fysiogr. sallskapet I, Lund Forhandlingar 33/J. Eickhoff, K. & R. Heipertz (1977): Discrimination of elevated immunoglobulin concentration in CSF due to inflammatory reaction 09 the central nervous system and blood-brain barrier dysfunction. Acta Neurol. Scand. 56, 475-482. Fahey, J. & E. M. McKelvey (1965): Quantitative determination of serum in macroglobulins in antibody-agar plates. J. Immunol. 94, 84. Fishman, R. A. (1975): The Nervous System, Donald B. Tower, Editor-in-Chief 2: The Clinical Neurosciences. Raven Press, New York. Ganrot,K. & C.-B.Laurel1 (1974): Measurement of IgG and Albumin Content of Cerebrospinal Fluid, and its Interpretatioa. Clin. Chem. 20, 571-573. Greenwood, B. M. & H. C. Whittle (1973): Cerebrospinal-fluid IgM in patients with sleeping sickness. Lancet ii, 525-527. Izikowitz, S. (1941): Studies on total protein in lumbar fluid. Diss. Stockholm, Elanders Boktryckeri. Jung, P. R., H. P. Rieder & C. Jeltsch (1973): Normalwerte des Totalproteins in Lumballiquor, Alters-Geschlechts- und Umweltabhangigkeit. Klin. Wschr. 51, 810-816. Laterre, E. Chr. (1973): The normal protein pattern in cerebrospinal fluid. La Ricerca in Clinica e in Laboratorio 3, 736. Laurell, C.-B. (1966): Quantitative estimation of proteins by electrophoresis in agarose gel-containing antibodies. Anal. Biochem. 15, 45. Laurell, C.-B. (1972): Electroimmunoassay. Scand. J. Clin. Lab. Invest. 29, Suppl. 124, 21-37. Link, H. & G. Tibbling (1977a): Principles of albumin and IgG analyses in neurological disorders. 11. Relation of the concentration of the proteins in serum and cerebrospinal fluid. Scand. J. Clin. Lab. Invest. 37, 391-396. Link,H. & G.Tibbling (1977b): Principles of albumin and IgG analyses in neurological disorders. 111. Evaluation of IgG synthesis within the central nervous system in multiple sclerosis. Scand. J. Clin. Lab. Invest. 37, 397-401. Mancini, G., A. 0. Carbonara & J. F. Heremans (1956): Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem. 2, 235. Mertin, E., H. Wisser & P. Doerr (1971): Untersuchung iiber den Normalbereich des Gesamt-Eiweisses und der Eiweissfraktionen des Liquor cerebrospinalis. Z . Klin. Chem. 9, 337-340. Rieder, H. P. & R. Wuthrich (1962): Die Cu-Folin-Methode zur Bestimmung des Gesarnteiweissgehaltes im Liquor. Klin. Wschr. 40, 1070-1075. Scheidegger, J. J. (1955): Une micromethode de l’immunoelectrophorese. In!. Arch. Allergy 7, 105. Teppo, A.-M. (1973): The 9th International Congress of Biochemistry, Stockholm, July 1-7. Abstract book.
365 Tibbling, G., H. Link & S. Ohman (1977): Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37, 385-390. Tourtellotte, W. (1959): Study of lipids in cerebrospinal fluid. Neurol. (Minneap.) 9. Weeke, B. (1961): Quantitative estimation of human immunoglobulins followings carbamylation by electrophoresis in antibody-containing agarose. Scand. J. Clin. Lab. Invest. 22, 107. Whittle, H. C. & B. M. Greenwood (1977): Cerebrospinal fluid immunoglobulins and complement in meningococcal meningitis. J. Clin. Path. 30, 720-722. Received July 19, accepted October 12, 1978
A . Ahonen, M.D.
Department o'f Clinical Chemistry University of Oulu SF-90220 Oulu 22
23 Acta neurol. scandinav. 58:6
Measurement of reference values for certain proteins in cerebrospinal fluid A. AHONEN, V. V. MYLLYLA AND E. HIOKKANEN Reference values are presented for certain cerebrospinal fluid proteins and related indexes, based on determinations carried out on 46 patients with diffuse lower back pain using radial immunodiffusion and electroimmunoassay. A sex difference was noted in total protein and albumin values. The electroimmunoassay method with its sensitivity enhanced by the use of Coomassie Blue staining and optimal antiserum concentrations enabled a reference value to be determined for IgA in cerebrospinal fluid. Key words: Cerebrospinal fluid proteins - electroimmunoassay - immunoglobulins - radial immunodiffusion - reference values
Despite its nonspecificity, the measurement of the total protein concentration in CSF is still regarded as useful in clinical practice, although an elevated protein content may be associated with a large number of quite disparate diseases. It has been suggested that this procedure should be replaced by CSF albumin determination, as measured CSF albumin levels vary much less from one laboratory to another than do values for total proteins, due to the more reliable methods and standards for calibration. Many other protein fractions of CSF have also been extensively discussed in the literature, but only one seems at present to have any diagnostic value, namely 7’globulin (principally the IgG portion), when its concentration is high (Fishman 1975). The colloidal reactions in common use earlier as indirect indicators of increased IgG concentration have been replaced by more specific techniques such as electroimmunoassay and radial immunodiff usion, which are also simpler for practical use than gel or filter paper electrophoresis. The purpose of the present work was to establish reference values for CSF total proteins, albumin and immunoglobulins and for certain indicative ratios, and to compare the results of radial immunodiffusion (RID) with those of electroimmunoassay (EIA).
359 PATIENTS AND METHODS The investigation was carried out on 27 men and 19 women at a clinic belonging to the Orthopedic Hospital of Invalid Foundation in Helsinki, Finland. They varied in age from 19 to 55 years with a mean of 39 years (mean 39 and median 41 for men, mean 38 and median 39 for women), and all had a history o t lower back pain for which they had been treated with various analgetic and antiinflammatory drugs and for which a lumbar myelography had been carried out. They included all the patients with negative findings in myelography or in operative (discus) exploration. There was no evidence or subjective complaint of any other neurological disease. Nor was evidence of lateral discus herniation obtained during the later follow-up time of 1 year. A routine lumbar puncture was carried out in a sitting or supine position and 2-3 ml of CSF was removed before injection of the contrast medium. A serum sample for similar laboratory tests was taken at the same time in 30 cases. The samples were stored immediately at -2OOC (CSF without centrifugation) and analyzed at the Kivela Hospital, Helsinki, Finland, within 2 to 6 weeks of sampling, during which period no significant alterations in protein levels were observed. Total protein concentration was measured by the routine biuret method, in which a c o b r complex binding between alkaline copper salt and protein is measured colorimetrically. Albumin and immunoglobulins were first determined by the standard RID method (Mancini et al. 1956, Fahey & McKeZvey 1965), using commercially available (Behringwerke, Marburg Germany) low concentration immunodiffusion agar plates for CSF analysis (PartigenQ. The second method involved immunoelectrophwesis (Sclzeidegger 1955, Laurel1 1966) using research grade EIA plates (ExaPhorB, Orion Diagnostica, Helsinki, Finland) further modified to obtain greater sensitivity and suitability for the present CSF investigations. EIA analyses were carried out oa a thin agar gel, which is a buffer substance also containing the antibody. The samples were first treated with ExaPhor formulant (formaldehyde) to change the electrical charges of the immunoglobulins to improve the migration (Teppo 1973) and after 10-20 min were placed in small circular wells punched in the gel layer. An electric field of 5 V/cm was then applied over the gel to incite electrophoretic migration of the antigen. The area of precipitate formed in the antigen-antibody reaction (rocket shape) is thus proportional to the amount of antigen in the sample (Weeke 1968, Laurel2 1972). The time needed for the formation of this precipitate was 3 h for albumin, 5 h for IgG and IgA and 6 h for IgM. The sensitivity of the plates for the detection of low concentrations of antigens in CSF was improved by Coomassie Blue staining and by optimizing the antiserum concentration, which showed to be 0.4-1.2 mg/l. The protein concentrations are expressed as milligrams per liter (mgll). The Student’s t-test and paired t-test were used.
RESULTS
The mean total CSF protein concentration (and SD) for the 46 patients was 363 k 70 mg/l (median 336) (Fig. la) that for the males being 378 k 69 mg/l and that for the females 337 66 m a . An almost significant difference ( P < 0.06) existed between the sexes, but no dependence on age could be derived from the present series. The CSF albumin and immunoglobulin levels are shown in Table 1. The respective albumin values were 187 k 58 mg/l (RID) 198 67 (EIA) and those for IgG 21.0 7.0 mg/l (RID) (Fig.
*
*
*
46 29 17 46 29 17 46 29 17 46 29 17
187 2011 166 21.0 20.6 21.8 5.7% 5.4 6.2 12.0 10.0 13.0
Means
11.3
5.9%
22.0
188
Medians 71-303 90-312 52-279 7-35 7-34 7-37 2.4-8.9 % 2.0-8.8 3.0-9.5 3.2-21.0 2.4-18.5 4.1-22.0
Reference values
Radial immunosdiffusion (RID)
46 29 17 46 29 17 302 13 17 30 13 17
No. of patients
P < 0.05 when compared with the corresponding value for the females. The lower number of patients in this group was due to small amount of CSF sample obtained.
Albumin males females IgG males females IgGItotal protein males females IgGIalbumin males females
No. of patients 198 205 186 25.0 24.3 26.1 5.2% 5.6 5.0 11.0 12.2 10.1
Means
10.8
5.3%
23
180
Medians
64-332 76-334 42-330 16-34 16-32 17-35 2.4-8.0% 3.6-7.6 1.8-8.2 2.3-20.1 7.0-17.3 0.0-2 1.2
Reference values
Electroimmunodiffusion (EIA)
Table 1 . Comparison of CSF albumin and immunoglobulin concentrations ( m g / l ) and various indexes immunodiffusion and electroimmunoassays. Means, medians and reference values (means & 2 S.D.)
0
o\
w
361
12
t .-LA--
36 39 MGlL
Figure 1 . Histograms f o r CSF total proteins ( a ) and IgG ( b ) concentrations of the present material.
l b ) and 25.0 2 4.3 mgA (EIA). The difference between the albumin values obtained by the two methods was not statistically significant, but the IgG concentrations obtained by RID were markedly lower than those given by EIA ( P < 0.01). The albumin values were lower in the females than in the males ( P < 0.05 with RID, P > 0.05 with EIA), but no significant sex differences were observed in the IgG concentrations. The ratios of IgG/total protein and IgG/albumin in CSF are also shown in Table 1. No statistical differences were observed here between the male and female patients. Correspondingly, Table 2 presents the CSF/serum ratios for IgG and albumin concentrations. Unfortunately EIA determinations could be carried out in this case on only eight patients, due to the small amount of sample taken. IgA and IgM concentrations in CSF were also measured in 12 of the patients using the same two methods, but the only detectable concentration was a reference value of 1.5 2 1.3 mg/l for IgA given by EIA.
362 Table 2. Comparison of CSF/serum ratios (promilles) f o r I g G and albumin obtained by radial immunodiffusion and electroirnmunoassay. Means and reference values (means k 2 S.D.)
RID No. of Means patients CSF-IgG/serurn-IgG males females CSF-albuminkerum alb. males females
30 12 18 30 12 18
1.7%0 1.6 1.8 5.0 4.8 5.1
EIA
Reference No. of values patients
Means
0.5-2.9%0 0.4-2.8 0.4-3.2 2.G8.0 2.0-7.6 2.1-8.1
8
1.7%0
0.7-2.7 550
8
3.7
2.0-6.0
Reference values
The reference values calculated from the present material are listed in Tables 1 and 2. Since reference values calculated by Student’s t-test and nonparametric tests did not noticeably differ from each other the former values were used. The skewness and kurtosis of the distribution of the results were statistically tested, but no significant difference from Gaussian distribution was observed. Two examples of histograms are presented in Fig. 1. DISCUSSION
Since for ethical reasons the authors believe that CSF samples should not be taken from healthy subjects, the present work was carried out entirely on patients with diffuse lower back pain in whom no evidence of herniation of an intervertebral disc could be verified in subsequent investigations (myelography or exploration and follow up). As the patients showed no signs of any other neurological disease, they may serve in our opinion as a “normal” reference group, although all minor abnormalities such as slight radiculitis or drug effects can not be excluded. The reference value of 223502 mg/l obtained here for CSF total proteins lies in the same range as those reported by Tourtellotte (1959) and Mertin et al. (1971), who used the biuret method. The almost significant differences found between the sexes by these authors seem to be due principally to differences in albumin levels, as can also be seen in the present material. The CSF total protein concentration was lower in the women than in the men, as also noted by Izikowitz (1941) and Dencker (1961, 1963), for example. On the other hand there are some investigators who have not found any sex difference in CSF protein levels (Rieder et al. 1962, Arnold 1966, Jung et al. 1973, Tibbling et al. 1977). This may be due to differences between patient groups, for it is known that protein levels in CSF are higher in older age groups than in younger ones, probably due to impaired function of the blood-brain barrier
363 (Rieder et al. 1962, Arnold 1966). The present material was not representative in that respect. Albumin is the largest protein fraction in CSF (cf. our reference value of 64-332 mg/l) and is formed outside the nervous system. It has been suggested that the CSF albumin level may give important information on CSF dynamics, with high levels referring to lesions of blood CSF barrier and/or reduced CSF turnover (Delpech & Lichtblau 1973, Ganrot & Laurell 1974). The sex differences appear to be clearer in the albumin values than in CSF total proteins, but the fact that the ratio between CSF and serum albumin levels is in the same range in both sexes suggests a dynamic equilibrium between the CSF and serum proteins. Since more reliable determination techniques are available, CSF albumin might well be preferred to total proteins. In addition, it would enable the CSF/serum ratio for albumin to be used as an index of blood-brain barrier function, in which even alterations in serum albumin levels will be taken into account (Delpech & Lichtblau 1973, Link et al. 1977a). The IgG concentration in CSF obtained here (735 mg/l) lies between the reference ranges reported by Glasner & Wenig (1973) and Ganrot & Laurell (1974) but lies in the same range as those given by Tibbling et al. (1977) and EickhofJ & Heipertz (1977) and shows no clear difference between the sexes. An elevated CSF/serum ratio of IgG is generally accepted as reflecting local IgG synthesis in the central nervous system (Ganrot & Laurell 1974), the usefulness of which seems to be proven in the diagnostics of multiple sclerosis, for instance. If simultaneous serum IgG values are not available, the local IgG synthesis in the central nervous system can be estimated from the ratio of IgG/total proteins or IgG/albumin in CSF (Delpech & Lichtblau 1973, Link et al. 1977b). No detectable concentrations of IgA and IgM could be shown by the RID method, and the EIA method gave a reference value for IgA, but not for IgM. Similar results have been reported by other investigators (Delank 1975, Laterre 1975), although IgM concentrations in CSF have been measured in pathological cases (Greenwood 1973, Whittle et al. 1977). The present results show that both methods are suitable for CSF albumin and IgG determinations. RID is easier to perform in routine work, but when very low concentrations, e.g. of IgA and IgM, are expected we would recommend ETA, which also has the advantage of requiring a shorter time. ACKNOWLEDGEMENTS The authors wish to thank Drs. K. Touru-Kaisila and K . Ojala for their assistance during the investigation. The Partigen plates were kindly donated by Hoechst Fennica, Helsinki, Finland and the ExaPhor electroimmuncrassay plates by Orion, Helsinki, Finland.
3 64 REFERENCES Arnold, F. (1966): Liquor-Untersuchungen an Gesunden und Multiple Sklerose-Kranken. Schweiz. Med. Wschr. 96, 1098-1103. Deland, H. W. (1971): Die Immunoglobuline im Liquor cerebrospinalis und ihr klinischdiagnostische Aussagewert. Nervenarzt 42, 483-490. Delpech, B. & E. Lichtblau (1973): Etude quantitative des immunoglobulines G et de l’albumine du liquide cCphalorachidien. Clin. Chim. 37, 15-23. Dencker, S. J. (1963): Studies on variation of CSF total proteins and cells with sex und age. Kungl. Fysiogr. sallskapet I, Lund Forhandlingar 33/J. Eickhoff, K. & R. Heipertz (1977): Discrimination of elevated immunoglobulin concentration in CSF due to inflammatory reaction 09 the central nervous system and blood-brain barrier dysfunction. Acta Neurol. Scand. 56, 475-482. Fahey, J. & E. M. McKelvey (1965): Quantitative determination of serum in macroglobulins in antibody-agar plates. J. Immunol. 94, 84. Fishman, R. A. (1975): The Nervous System, Donald B. Tower, Editor-in-Chief 2: The Clinical Neurosciences. Raven Press, New York. Ganrot,K. & C.-B.Laurel1 (1974): Measurement of IgG and Albumin Content of Cerebrospinal Fluid, and its Interpretatioa. Clin. Chem. 20, 571-573. Greenwood, B. M. & H. C. Whittle (1973): Cerebrospinal-fluid IgM in patients with sleeping sickness. Lancet ii, 525-527. Izikowitz, S. (1941): Studies on total protein in lumbar fluid. Diss. Stockholm, Elanders Boktryckeri. Jung, P. R., H. P. Rieder & C. Jeltsch (1973): Normalwerte des Totalproteins in Lumballiquor, Alters-Geschlechts- und Umweltabhangigkeit. Klin. Wschr. 51, 810-816. Laterre, E. Chr. (1973): The normal protein pattern in cerebrospinal fluid. La Ricerca in Clinica e in Laboratorio 3, 736. Laurell, C.-B. (1966): Quantitative estimation of proteins by electrophoresis in agarose gel-containing antibodies. Anal. Biochem. 15, 45. Laurell, C.-B. (1972): Electroimmunoassay. Scand. J. Clin. Lab. Invest. 29, Suppl. 124, 21-37. Link, H. & G. Tibbling (1977a): Principles of albumin and IgG analyses in neurological disorders. 11. Relation of the concentration of the proteins in serum and cerebrospinal fluid. Scand. J. Clin. Lab. Invest. 37, 391-396. Link,H. & G.Tibbling (1977b): Principles of albumin and IgG analyses in neurological disorders. 111. Evaluation of IgG synthesis within the central nervous system in multiple sclerosis. Scand. J. Clin. Lab. Invest. 37, 397-401. Mancini, G., A. 0. Carbonara & J. F. Heremans (1956): Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochem. 2, 235. Mertin, E., H. Wisser & P. Doerr (1971): Untersuchung iiber den Normalbereich des Gesamt-Eiweisses und der Eiweissfraktionen des Liquor cerebrospinalis. Z . Klin. Chem. 9, 337-340. Rieder, H. P. & R. Wuthrich (1962): Die Cu-Folin-Methode zur Bestimmung des Gesarnteiweissgehaltes im Liquor. Klin. Wschr. 40, 1070-1075. Scheidegger, J. J. (1955): Une micromethode de l’immunoelectrophorese. In!. Arch. Allergy 7, 105. Teppo, A.-M. (1973): The 9th International Congress of Biochemistry, Stockholm, July 1-7. Abstract book.
365 Tibbling, G., H. Link & S. Ohman (1977): Principles of albumin and IgG analyses in neurological disorders. I. Establishment of reference values. Scand. J. Clin. Lab. Invest. 37, 385-390. Tourtellotte, W. (1959): Study of lipids in cerebrospinal fluid. Neurol. (Minneap.) 9. Weeke, B. (1961): Quantitative estimation of human immunoglobulins followings carbamylation by electrophoresis in antibody-containing agarose. Scand. J. Clin. Lab. Invest. 22, 107. Whittle, H. C. & B. M. Greenwood (1977): Cerebrospinal fluid immunoglobulins and complement in meningococcal meningitis. J. Clin. Path. 30, 720-722. Received July 19, accepted October 12, 1978
A . Ahonen, M.D.
Department o'f Clinical Chemistry University of Oulu SF-90220 Oulu 22
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