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.

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.

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.

Isoelectric focusing of CSF and serum proteins in neurological disorders combined with benign and malignant proliferations of reticulocytes, lymphocytes and plasmocytes.

The clinical data and the CSF and serum findings on isoelectric focusing (IEF) and electrophoresis were studied in nine patients with neurological disorders combined with proliferations of reticulocytes, lymphocytes or plasmocytes. Paraneoplastic neurological manifestations were considered in some of the subjects. CSF findings compatible with intrathecal synthesis of oligoclonal immunoglobulins were observed in two patients with lymphoreticular neoplasms. One of these had an IEF band spectrum differing from those found with multiple sclerosis (MS), while the other had changes indistingusihable from those with MS. On IEF the CSF and serum M components of the patients with plasma cell dyscrasias, as well as the serum M components of nine other patients with myelomatosis, exhibited considerable microheterogeneity with 3-18 abnormal bands. The isoelectric point (pI) of the individual bands was 5.8-9.2, mostly greater than 6.4; the band spectra exhibited pI ranges of 0.4-2.5 pH units, most frequently 0.4-1.2. The CSF findings, possible modified by the blood-CSF barrier, reflected the serum changes of plasma cell dyscrasias. The immunoglobulins produced in such disorders gave IEF band spectra differing from those of intrathecally produced immunoglobulins in MS.

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.

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.

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.

Isoelectric focusing and electrophoresis of the CSF proteins in tremor of different origins.

The CSF proteins have previously been very little investigated in the cerebellar syndrome of chronic alcoholism and in essential tremor. Such studies have been carried out more thoroughly by electrophoretic methods in Parkinson's disease but generally with normal results. In the present investigation the CSF proteins were examined by isoelectric focusing and quantitative paper electrophoresis in 10 patients with the cerebellar syndrome of chronic alcoholsm, 12 patients with Parkinson's disease and 16 subjects with essential tremor. Abnormal CSF proteins of very similar appearance were found on isoelectric focusing in the acidic pH interval 5.6-5.8 in 80% of the patients with the cerebellar syndrome of chronic alcoholism. In Parkinson's disease the most common aberration was evidence of nonspecific blood-CSF-barrier damage which occurred in half of the patients. In only 17% of these cases did other alterations appear, situated in the pH range alkaline to pH 5.8. Abnormal CSF proteins were found in 94% of the patients with essential tremor. The aberrant proteins appeared in both the acidic and alkaline pH regions, most frequently with anisoelectric point at pH 5.9, 7.2 and 9.3. There was a considerably higher frequency of CSF protein abnormalities in different pH ranges in patients with tremor of more pronounced degree as compared to those with only mild symptoms. The electrophoretic examinations failed to show any conclusive alterations. Barrier-damage patterns of mild or moderate degree or slightly increased levels of CSF beta1-globulin were occasionally found in all 3 diseases. The results indicate that isoelectric focusing of the CSF proteins may be of diagnostic value in the cerebellar syndrome of chronic alcoholism and in essential tremor but does not reveal any characteristic abnormalities in Parkinson's disease.

Isoelectric focusing and electrophoresis of cerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies.

In the very few previous investigations of the CSF-proteins in muscular dystrophies the results have generally been reported as normal. In spinal muscular atrophies a barrier-damage pattern of CSF-proteins has been found in amyotrophic lateral sclerosis (ALS). In the present investigation the CSF-proteins were examined by isoelectric focusing and quantitative paper electrophoresis in 13 patients with muscular dystrophies and in 11 patients with spinal muscular atrophies. On isoelectric focusing, CSF-protein abnormalities were found in 85% of the cases with muscular dystrophies and in all patients with spinal muscular atrophies. Differences in the CSF-protein patterns were observed within the group of muscular dystrophies and between these and the cases of spinal muscular atrophies. In ALS and in myotonic dystrophy, abnormal CSF-protein fractions occurred mainly in the alkaline pH-range, while in limb-girdle dystrophy and the patient with facioscapulohumeral dystrophy, aberrant fractions appeared mainly in the acidic region. CSF-protein abnormalities were found in both the alkaline fractions (HAFs) with pI 9.2-9.6 and a fraction with PI 7.1 were found in half of the patients with myotonic dystrophy. The CSF electrophoresis in myotonic dystrophy showed increased levels of beta1-globulin in all cases examined. Signs of barrier-damage were commonly encountered in ALS in contrast to the muscular dystrophies, except for myotonic dystrophy. The results are discussed in terms of possible diagnostic value and with regard to pathogenetic significance, particularly in relation to the current hypothesis of a neural involvement in muscular disorders.

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.

Cerebral haemorrhages with atypical clinical patterns. A study of cerebral haematomasusing CSF spectrophotometry and computerized transverse axial tomography ("EMI scanning").

Twelve patients with 13 cerebrovascular incidents presenting with atypical clincial patterns where thecombination of CSF spectrophotometry (CSF-SPE) and computerized transverse axialtomogrpahy(CTAT, "EMI-scanning") showed cerebral haematomas, with or without haemorrhage into the CSFare reported. The high diagnostic significance of SDF-SPE as well as the value of using a combination of CSF-SPE and CTAT examinations in cerebrovascular disease has preveiouslybeen established by the authors. The present patient group comprised some 10% of a totalseries of patients with cerebrovascular disease hitherto studied in this fashion. Conclusive findings were found in only 1 fo the 5 cases examined by angiography and in none of the5 cases studied by isotope scan. Since many cerebral haemorrhages obviosly haveatypical clincalfeatures as compared with the "classical" pattern, the combined use of CSF-SPE and CTAT examinations in the diagnosis of cerebral haematomas is a prereguisite for rational therapy.

Protein patterns of cerebrospinal fluid in hereditary ataxias and hereditary spastic paraplegia.

The CSF findings in hereditary ataxias and allief disorders have hitherto mostly been reported as normal if one excludes Refsum's syndrome. The CSF-protein patterns found on isoelectric focusing and quantitative paper electrophoresis were studied in 12 patients with hereditary ataxias and hereditary spastic paraplegia. Using a recently-developed technique of isoelectric focusing of CSF-proteins in flat beds of polyacrylamide gel, the authors could show abnormal CSF-protein patterns in all but 1 of the present cases. The aberrant CSF-protein patterns found showed differences between the syndromes studied. Two unique patterns with conspicuous fractions in the acid range were observed in patients with Marie-Sanger-Brown's ataxia (mother and daughter) and Holmes' ataxia. A third CSF-protein pattern was found in a sibship with Friedreich's ataxia including a double fraction in the acid region (pI 5.9-6.1) in all 4 subjects and a highly alkaline fraction (HAF) with pI about 9.3, in 3 of them. Similar acid fractions (pI 5.9-6.1) were also detected in 3 of 4 patients with hereditary spastic paraplegia, a brother and sister showing a very similar CSF-protein pattern. Double fractions with pI 5.9-6.1 and/or HAF may also occur in other neurological diseases, mostly, however, associated with other distinctive features of their CSF-protein patterns. A possibility in the future of distinguishing hereditary CNS-diseases by examination of the CSF-protein pattern is suggested.

Analytical isotachophoresis: a new method for analysis of cerebrospinal fluid proteins. A preliminary report.

Isotachophoresis of CSF proteins seems to be a very promising method. Very small CSF samples, a few mul of concentrated and 15-30 mul of unconcentrated CSF, can be quickly analysed (30-60 min), and the results immediately obtained on a recorder. Using unconcentrated CSF, losses due to concentration procedures, are avoided. Low-molecular weight compounds, e.g. in CSF ultrafiltrates, can also be examined. The method gives high resolution, is reproducible, and is easy to perform. The isotachophoretic findings have been compared with those of electrofocusing.

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.

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).

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.

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.