RESUMO
The following method is proposed to make the serum concentration of the middle molecules evident and to isolate them: 1) Fractioning of serum by gel filtration with sephadex G 75 columns in order to eliminate proteins and other substances with mol. weight less than 3,000-3,500. 2) Collection of fractions with mol. weight greater than 3,000-3,500 their lyophilisation and their successive gel filtration by sephadex G 15 columns. The concentration of substances in the single fractions is evaluated by ultraviolet light absorption (wave length 254 nm). The peaks of the patterns show a great difference between normals and uremics. The uremic peaks are always higher than normals. The mol. weight the substances in the peaks obtained by gel filtration with sephadex G 75 and G 15 was verified by radiolabelled substances of known mol. weight added before gel filtration. The substances isolated by sephadex G 75 from 10 and 20 cases of uremics and normal controls were diafiltered by Diaflo membrane UM 05 (nominal mol. weight cut - off 500). The diafiltered substances (mol. weight between 500 and 3,500) were investigated. A great difference between uremics and normal controls of dry mol. weight was observed, while the percentage concentration of C, H, N and of amino acids evaluated after acid hydrolysis showed no difference between the two groups. The liberation of amino acids after acid hydrolysis confirmed the presence of peptides among the middle molecules. Middle molecules in the urine of uremic and normal subjects have been investigated by the same method.
