Microdialysis of cerebrospinal fluid in polyacrylamide gels: a suitable procedure prior to electrophoretic analysis.

Most electrophoresis methods for separation of CSF proteins are generally preceded by some procedure of concentration and desalting of the specimen. Generally ultrafiltration techniques are used. The risk of losses, which may be unequal for different CSF proteins during such procedures, is to be stressed. On the other hand, desalting prior to isoelectric focusing (IEF) will minimize the curvature of the protein bands, and in isotachophoresis (ITP) faster separation and increased capacity with repeated sample application are made possible. Since some years microdialysis of samples has been performed by the authors and found to be a valuable procedure both prior to IEF and ITP. With respect to microheterogeneity and recovery, tested by IEF, immunonephelometry and radioiodinated proteins no losses were observed. Ion exchange of acrylamide in a mixed resin, and recrystallization of bisacrylamide, were found necessary to avoid absorption from very dilute protein solutions as CSF. Gel structure and performance were very dependent on polymerization conditions (time, temperature, initiator and accelerator concentrations).

Benign monoclonal gammopathy and peripheral neuropathy.

Peripheral neuropathy has been described in malignant plasma cell dyscrasias such as multiple myeloma and Waldenstöm's macroglobulinaemia. Since it is not known whether the neuropathy is related to the plasma cell disorder or is a paramalignant phenomenon, 21 consecutive out-patients with benign monoclonal gammopathy (BMG) were analysed for peripheral neuropathies. Eleven patients had noticed slight motor and/or sensory extremity symptoms. Clinical examination, electromyographic and electroneurographic studies of the upper and lower extremities were performed. In five patients all results indicated a neuropathy, six other patients had clinical signs of neuropathy and four additional patients had positive electromyographic and/or electroneurographic results compatible with neuropathy. There were no significant differences in haematological parameters between the group where all results indicated a neuropathy and the totally negative group or between the two groups with and without clinical neuropathy. Thus, the benign form of plasma cell dyscrasias seems also to be associated with mild clinical or subclinical peripheral neuropathy.

Peripheral neuropathy in patients with benign monoclonal gammopathy--a pilot study.

It is well known that peripheral neuropathy occurs in patients with myeloma or macroglobulinaemia, but its pathogenesis is still obscure. In recent years, neuropathy has also been reported in association with benign monoclonal or oligoclonal gammopathy. Modern histo-immunological methods have revealed evidence of antibody production to peripheral nerve tissue, probably the myelin sheath. The present study included 21 unselected, consecutive patients with benign monoclonal gammopathy observed in the Division of Haematology. Clinical and laboratory investigations included electrophysiological examination and analyses of the M components. Of the 21 patients 11 had noticed slight neuropathic symptoms in their extremities; in 5 both clinical and electrophysiological findings were compatible with neuropathy; 6 showed positive clinical signs of neuropathy; 4 had either positive electromyographic or electroneurographic findings. In summary, 15 of 21 patients had some signs of peripheral neuropathy. In spite of the screening design of the study, this strikingly high frequency is comparable with other recent reports. Haematological studies did not reveal any significant differences between the patient groups with positive or negative neurological findings. The findings indicate that even benign gammopathies may be associated with peripheral neuropathy.

High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum evaporation technique before IF.

Effects of bezafibrate on low HDL-cholesterol in patients with ischaemic cerebrovascular disease: a pilot study.

Bezafibrate is a new lipid-lowering agent that quite constantly increases low HDL-cholesterol values in hyperlipoproteinaemic patients. The possible role fo HDL-cholesterol as an anti-atherogenic factor has been frequently discussed, mainly in patients with ischaemic heart disease but recently also in ischaemic cerebrovascular disease (ICD). This is the first pilot study in six selected patients suffering from ICD who had at the same time low HDL-cholesterol values (less than or equal to = 1.1 mmol/l) with otherwise normal lipids. After a wash-out period of 2 months duration these patients were treated with 200 mg bezafibrate t.i.d. for 2 months. They were then followed up for another 8 months. Bezafibrate therapy increased HDL-cholesterol (range 45-130%). Eight months after cessation of therapy five patients have returned to pathologically low HDL-levels and the sixth patient also has a relatively low value of 1.2 mmol/l. This small preliminary study cannot, however, provide evidence about the possible beneficial role of increasing HDL-cholesterol in patients with ICD. Further investigations are therefore in progress.

Isoelectric focusing of cerebrospinal fluid proteins in ischemic cerebrovascular disease.

The cerebrospinal fluid (CSF) protein patterns, in ischemic cerebrovascular disease (ICD), of varying extension were studied by isoelectric focusing (IEF) in 100 patients at different intervals after the onset of symptoms. The diagnoses were based on conventional clinical examinations and CSF spectrophotometry in all cases. Computed tomography was performed on 52 cases. One or more CSF protein aberrations were noted in 94 patients. Some of these findings were most common with small lesions including TIA. Other aberrations were most frequent with the more extensive infarctions. A regional increase in the gammaglobulin range was found in six cases. The findings were most frequent in the first days after the stroke except for barrier damage which reached a maximum during the second week. The IEF findings of CSF seem to be of diagnostic value. Taken together with the clinical signs and CT findings, they could conceivably give prognostic information.

Neuropathies associated with disorders of plasmocytes.

Neurological syndromes, e.g. polyneuropathies, can be associated with abnormal production of immunoglobulins, as for example in multiple myeloma. The pathogenetic mechanisms for the development of neurologic symptoms have often, indeed, remained obscure and speculative. This article presents some aspects of gammopathies correlated to polyneuropathy. Results of protein analyses of plasma and cerebrospinal fluid, utilizing a high separation technique, are presented. We also introduce a few case histories, illustrating the possible association between polyneuropathy and benign monoclonal gammopathy, hitherto regarded as an abnormality without clinical significance. The possible association between immunoiglobulin findings in cases of polyneuropathy with unknown etiology and a good response to steroid therapy is also mentioned.

Determination of CSF proteins by a simple and rapid immunonephelometric method.

A simple, fast and reliable immunonephelometric (I-NEPH) method has been worked out for the determination of CSF proteins. A good quality and low price I-NEPH apparatus was used. The results were obtained from regression lines, constructed from various parts of the calibration curve, calculated by using a simple pocket calculator. Ultrasound was found to be a simple and effective cleaning technique for nephelometry. The method was used for determining concentrations of albumin, IgG, IgA, IgM and the ratio of the light kappa and lambda chains in CSF and serum from 15 control cases, 11 MS patients, and three patients with plasma cell dyscrasias. The I-NEPH method was found to be a valuable complement to high separation techniques including especially isoelectric focusing used for CSF examinations, e.g., for evaluating influence of serum protein composition and degree of barrier damage. An increased kappa-lambda ratio was observed in some of the patients with MS in accordance with previous investigations but was normal in four of the nine MS cases where the ratio was examined.

Isotachophoresis of CSF proteins in gel tubes especially gammaglobulins. An analytical and preparative technique for high-separation of CSF proteins.

An isotachophoretic method using polyacrylamide gel (PAG-ITP) in a simple disc electrophoretic equipment with plastic tubes containing the gels, was elaborated and especially designed for studying the gammaglobulins in CSF and serum from control subjects and patients with neurological disorders, especially known or probable MS. The device and the ITP system used, including leading and terminating electrolytes and spacer substances, dividing the gammaglobulins in a reproducible way, are described. No cooling of the gel tubes was needed. The sample volumes varied between 5--500 microliters, and the separation time was 1.5--3.0 h. CSF from patients with verified or probable MS revealed characteristic, increased low-mobility gammaglobulin fractions. Using other ITP systems, such as other spacer compositions, the anodic proteins can also be studied in more detail. PAG-ITP in gel tubes is a simple and inexpensive technique which can be used for both analytical and preparative procedures for biological material such as CSF, serum and extractions from nervous tissues.

Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

Isoelectric focusing of CSF proteins in known or probable infectious neurological diseases and the Guillain-Barré syndrome.

The CSF and serum proteins of 120 patients with known or probable infectious neurological diseases or the Guillain-Barré syndrome were examined with thin-layer IEF. All but two of these patients exhibited one or combinations of different CSF-protein aberrations in the acidic and alkaline range. Aberrant non-Ig fractions (including transferrin, the tau-fraction and gamma-trace protein) were found in frequencies varying between 4 and 48%. CSF Ig components of restricted heterogeneity, i.e. oligoclonal bands and/or regional increases of gamma-globulins, were more frequent in patients with (meningo-)encephalitic or (meningo-)-myelitis/radiculitic disorders (respectively 69 and 48%) than in subjects with meningitis or Guillain-Barré syndrome (17%). The occurrence of such Ig abnormalities was higher in subacute or chronic than in acute disease and in subjects examined greater than 4 weeks after the onset rather than earlier. Ig-band spectra with marked anodal extension were found predominantly in (meningo-)encephalitic disorders with infratentorial symptoms. Age and sex were not found to influence the occurrence of abnormal Ig fractions. Such components could be detected in spite of pronounced blood-CSF barrier defects.

CSF protein examinations with thin-layer isoelectric focusing in multiple sclerosis.

Thin-layer IEF, due to its extremely high resolving capacity, has been found to be quite valuable for CSF protein examinations, one important advantage of the technique being its excellent capacity for separation of immunoglobulins. The CSF and serum proteins of 230 patients with clinically verified or probable MS and 20 subjects with optic neuritis were examined with thin-layer IEF and the findings were compared with clinical data and results of other CSF examinations. All but 3 of the MS patients and about two thirds of the subjects with optic neuritis inhibited one or combinations of different CSF protein aberrations in the acidic and alkaline range. Oligoclonal bands and/or regional increases of Ig fractions, changes compatible with intrathecal Ig synthesis, were detected in respectively 95 and 80% of patients with clinically verified and probable MS and 30% of subjects with optic neuritis. Other aberrant CSF protein fractions (including transferrin, the taufraction and gamma-trace protein) were found in about half of the cases; some of these fractions had the highest occurrence in patients with the most extensive Ig abnormalities. The diverse CSF protein aberrations seemed to be influenced by the duration and course of the disorder as well as the probable sites of lesions; further factors might be the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the presence of possible agents.

Dyslipoproteinemia in patients with ischemic cerebro-vascular disease: a study of stroke before the age of 55.

Serum lipoproteins were determined 8-12 weeks after the onset of ischemic cerebro-vascular disease (ICD) in 61 patients, 38 males and 23 females, before the age of 55. The results were compared with those of a matched control material. The diagnosis was based on clinical findings, CSF spectrophotometry, computer tomography, and angiography. Hyperlipoproteinemia was no common finding in these young and middle-aged patients with ICD. The normal mean total serum cholesterol concentration was the result of a slight increase in VLDL cholesterol and a concomitant HDL cholesterol reduction. In men, the HDL cholesterol concentration was lower than expected for any VLDL-TG concentration. The mean value of the HDL cholesterol concentration in the patients was 18% lower than in the control group. On agarose electrophoresis the lipoprotein variants "late prebeta", "sinking prebeta" and "rapid beta" lipoproteins could be demonstrated in the same frequency as in controls. There was no significant correlation between the degree of atherosclerosis, estimated by angiography, and any serum lipoprotein fraction. Several recent studies have stressed the importance of a low HDL concentration as an independent risk factor for atherosclerosis. The decreased HDL cholesterol levels found in the present material require further attention to the possible beneficial role of HDL in ICD.

Spectrophotometry of the CSF (CSF-SPE) and computer tomography (CT) in traumatic head injuries.

Combined examinations with quantitative CSF spectrophotometry (CSF-SPE) and computer tomography (CT) were performed on 53 patients with traumatic head injuries. In cerebral concussion the results were mainly normal in both examinations. In cerebral contusion bleeding patterns were found by CSF-SPE in all subjects, with a special bleeding pattern (S2 pattern) occurring in 86%. CT showed findings described as typical for contusion in 8 of 14 examined patients, the remaining CT scans showing questionable or normal signs. In extra- and intracerebral haematomas, all patients had bleeding patterns on the CSF-SPE. A special bleeding component (H factor) was found in about 72%. The H component was not observed during the first 3 to 4 days after the trauma. All but one patient examined later than the 4th day had an H component with or without an S-pattern. CT demonstrated a haematoma in 14 of 18 verified haematoma patients, while 4 subjects with subdural haematoma (e.g. one third of this group) had questionable CT findings. The combined examinations with CT and CSF-SPE, being complementary to each other, are of great value in the different diagnosis of traumatic head injuries.

Normal serum-cholesterol but low H.D.L.-cholesterol concentration in young patients with ischaemic cerebrovascular disease.

Serum-lipoproteins were determined in male and female patients aged under 55 who had survived an attack of ischaemic cerebrovascular disease (I.C.D.). The results were compared with findings in healthy controls. Total serum triglyceride and cholesterol concentrations were not increased. However, in the very-low-density lipoprotein fraction increased cholesterol concentrations were found, and the mean value of high-density-lipoprotein (H.D.L.) cholesterol in I.C.D. patients was 18% lower than in controls. Since a low H.D.L.-cholesterol concentration has been suggested as an independent strong risk factor, it is possible that the susceptibility of I.C.D. patients to atherosclerosis is the result of a low H.D.L. rather than hyperlipoproteinaemia.

Isoelectric focusing and isotachophoresis for investigation of CSF and serum proteins in demyelinating and infectious neurological diseases.

Isoelectric focusing (IEF) and isotachophoresis (ITP), two methods with excellent separation capacities, have been adapted during recent years for the analysis of CSF proteins. The fractions separated by these techniques can be further studied by e.g. immunological methods. ITP has besides its high separation capacity several valuable advantages: very small samples are needed, unconcentrated CSF can be examined, the analyses are quickly performed and the results are immediately obtained on a recorder. Examinations by thin-layer IEF in a series of about 2,000 patients have afforded much new information about the CSF and serum proteins in many neurological diseases. Different complex CSF protein aberrations have been found in the gammaglobulin range as well as in more anodal positions in MS, infectious neurological diseases and Guillain-Barré syndromes. These aberrations are probably the result of several interacting factors, e.g. the temporal and spatial characteristics of the disease, the release of decomposition products from destroyed tissues, the genetically determined reactivity of the individual and the type of etiological agent.