Quantitative isolation and purification of blood group-active glycosphingolipids from human B erythrocytes

11.4 mg of a ceramide hexahexoside (B-I) and 16.4 mg of a ceramide octahexoside (B-II) as blood group B-active glycosphingolipids composed of glucose, galactose, N-acetylglucosamine and fucose (molar ratios 1:3:1:1 and 1:4:2:1 respectively) have been isolated from 6,400 ml of packed human B erythrocytes. This yield is greater by more than the 13-fold amount of B-I glycosphingolipid and the 20-fold amount of B-II glycosphingolipid which has hitherto been isolated from human erythrocytes. The B-active glycosphingolipids isolated represent about 0.04% of the erythrocyte membrane and in consequence must be regarded as the main representants of B properties of the erythrocytes as far as they have been investigated up to this time. This high yield was achieved by a simple and conservative erythrocyte membrane preparation without loss of serological activity and by the improvement of some chromatographical methods which permitted a high purification without the acetylation-deacetylation procedure. Purity was checked by gas-liquid chromatographical analysis of the sugars as their alditol acetates and by the hemagglutination inhibition technique. 1.7 x 10-8 g of each of these glycosphingolipids completely inhibit the agglutination of human B erythrocytes by 4 hemagglutination units of normal human anti-B sera. A ceramide tetrahexoside and a glycolipid fraction with a high H activity could also be isolated which possibly are blood group-intermediate substances. Lewis blood group-active glycosphingolipids characterized by the hemagglutination inhibition test and by passive hemagglutination are trace constituents of other glycosphingolipid fractions.