Heating unsaturated fatty acids in air produces hemagglutinins

When mono-unsaturated fatty acids are heated in air, they form hemagglutinins. When the double bond is Δ-6,7 or Δ-9,10, the titer is higher than for Δ-11,12. Stearic acid does not become a hemagglutinin on heating. Hydroxy-monounsaturated fatty acids, ricinoleic (cis-12-OH-Δ-9) and ricinelaidic (trans-12-OH-Δ-9) are not hemagglutinins unless they are heated. Oleic acid (Δ-9-octadecenoic acid, OA) has a very low agglutination titer but lyses red cells at higher concentrations. Rabbit and rat erythrocytes (RBC) give the highest titers but RBCs of other species are also agglutinated. OA was chosen for further study. Its specific titer against rat RBCs increases with time of heating in air. Thin-layer chromatography (TLC) and mass spectroscopy (MS) show that higher molecular weight compounds are formed and that activity is associated with these materials. Synthetic (oxidation of oleic acid with tert-butyl peroxide) and commercial preparations of oleic acid dimers (Emery and Unichema) and a commercial preparation of oleic trimer mixed with polymer (Emery) have high hemagglutination titers against rat erythrocytes. A cyclic, long-chain dicarboxylic acid, 5(6)-carboxy-4-hexyl-2-cyclohexene-1-octanoic acid (Westvaco) gives a very low titer unless heated and no lysis. Sialidase treatment of the red cells increases the titer. Removal of cations does not alter the titer but addition of Ca2+ or Mg2+ lowers the titer. Light microscopy was used to characterize and visualize the agglutination process with rat RBCs. Agglutination without lysis or fusion is observed for low concentrations of heated oleic acid and C-18 dimers and trimer-polymer preparations, and no large vesicles are seen. We conclude that the oligomeric fatty acids with two or more hydrophobic chains of seven or more carbons are agglutinins at physiological pH. Agglutination by dimer may be the result of the its two hydrophobic side chains inserting into adjacent RBC membranes or the result of dimer inserting completely into RBC membranes and altering their properties. The carboxyl groups may also play a role in the process by interacting with polar headgroups in the RBC membrane.