Determunation of cerebrospinal fluid protein and their fractionation by isoelectric focusing
The cerebrospinal fluid is a water-clear ultrafiltrate of plasma contained in the ventricular and the subarachnoid spaces of the brain and spinal cord. It is formed mainly by a process of secretion from villous projections of the choroid plexuses in the ventricles of the brain. The total volume of cerebrospinal fluid in a healthy, adult human is about 130ml. Physical properties such as osmotic pressure, pH and hydrostatic pressure are the same as in plasma, however, as an ultrafiltrate it differs from plasma in having no bilirubin, less sugar, calcium, nonprotein
nitrogen, and extremely low protein, but more chloride and about the same concentration of bicarbonate.
Electrophoresis is an useful technic for studying the cerebrospinal fluid proteins in health and pathological states. Various technics for concentrating cerebrespinal fluid prior to electrophoretic separation of protein fractions were tested by many investigators. These included pressure ultrafiltration through protein impermeable membranes, dialysis against concentrated solutions of polymers, and protein precipitation by means of acetone. Kaplan and Johnstone (1966) reported that the proteins of cerebrospinal fluid, which were concentrated by the vacuum ultrafiltration method, were separated into 6-7 fractions on the cellulose acetate electrophoresis. E$vans and Quick (1966), used polyacrylamide disc electrophoresis have reported results of 15 bands of protein in cerebrospinal fluid which were
applied in gel following concentration by pressure dialysis. Cunnningham (1964) published a series of five example of cerebrospinal proteins separated by acrylamide disc gel electrophoresis and using a native or unconcentrated sample
mixed with sucrose and layered over a gel column, a minimum of 22 bands were separated from the fluid examined.
Isoelectric focusing which is considered to be relatively new technique in recent years is the most sensitive and has an advantage to choose the empholyte of appropriate pH ranges. Isoelectric focusing involves the application of a potential
difference between the ends of a column of electrolyte in which there is a pH gradient.
In this study cerebrospinal fluid was obtained by atraumatic lumbar from 22 hospitalized patients with no evidence of neurological disease. The protein of cerebrospinal fluid was determined by micro-Kjeldahl method and their fractionation
was separated by means of isoelectric focusing. Double diffusion in agar-gel was performed according to modified Ouchterlony (1958) method.
The results obtained are summarized as follows:
Total protein concentration of cerebrospinal fluid was 29.8±3.59 mg/100ml. By isoelectric focusing, using Ampholine carrier ampholyte pH 3-10 formed in a thin layer of polyacrylamide gel in which sample were mixed homogenously, nineteen fractions were identified and most of them appeared to have low isoelectric point. Antigenecity of protein in cerebrospinal fluid was same as serum against antihuman rabbit serum.
By the above results, it may be concluded that the albumin occupied most part of components in protein of cerebrospinal fluid.