The lipids of fish. 7. Phosphate esters in the lipids of haddock and cod flesh

Abstract

Phospholipid fractions from haddock and cod muscle, containing appreciable quantities of unidentified lipids, were hydrolyzed with 0.5 N KOH in 95[degree] ethanol for 2 hours under reflux. K salts of phosphate esters from these hydrolysates were fractionated on the basis of their solubility in ethanol, by paper chromatography, by stability to hydrolysis at pH 4 for 7 hours in an autoclave at 15 lb. pressure and by stability to hydrolysis at pH 4 after a preliminary hydrolysis with N HC1. These methods showed the presence of a series of phosphate esters based on glycerol and small quantity of phosphoric acid in some other combination. A micro-chromatographic method for the estimation of glycerol was developed, whereby amounts of glycerol and phosphoric acid liberated under different conditions of hydrolysis could be correlated. Phosphate esters with more than 1 glycerol residue/P-atom were found in the ethanol-soluble fractions, giving evidence of bisphosphatidic acid-type structures. These could be divided into 2 classes according to stability at pH 4. Acid-periodate oxidation was completely unspeci- fic for glycerophosphate esters. In some fractions acid-periodate oxidation liberated more phosphoric acid than was formed by hydrolysis at pH 4, which is known to yield quantitative hydrolysis of glycerophos-phoric acid. In other fractions, hydrolysed with N HCl and then at pH 4, the glycerol and phosphoric acid liberated were more than could be accounted for by the results obtained from acid-periodate oxidation. The dangers of using even weakly acid ion exchange resins for the separation of phosphate esters were discussed.