Choline-containing phosphatides separation

Taurog and his associates (174) found that phosphatides are absorbed quantitatively from a petroleum ether solution by magnesium oxide 0.6 g. magnesium oxide suffices to absorb 0.85 mg. of phosphatide phosphorus dissolved in 15 ml. petroleum ether. Use of 1.75 g. magne.sium oxide is however preferable when choline-eontaining phosphatides have to be separated from non-choline-containing phosphatideS. The separation is based on the elution of the choline-containing phosphatides by methyl alcohol. 

The separation of choline-containing phosphatides from non-choline-containing phosphatides can however also be accomplished by first dissolving the phosphatides in methanol and then treating this methanol solution with magnesium oxide. The following procedure is used. 

In the work-up of the diethyl ether reaction, the solvent is removed (in a hood) under a stream of nitrogen. Then the ether-free aqueous dispersion is mixed with 10 ml of a 5 mM EDTA solution to bind the calcium ions. Without the latter treatment, the calcium salt of the phosphatidic acid cannot be recovered (easily) from the reaction mixture. Thus, subsequent to the EDTA treatment, the sample is mixed with sufficient chloroform and methanol to make a mixture of chloroform-methanol-water (1 2 0.8, v/v). Then 0.5 volume chloroform and 0.5 volume water are added, and the mixture is vigorously shaken for 1 min and then allowed to separate into two phases. The upper, water-rich phase contains free nitrogen base—in this case, choline. It can be analyzed by the techniques described earlier. 

Lecithin, a mixture of phosphatidyl choline, phosphaddyl ethanolamine, and phosphatidyl inositol, is used as a surfactant in many food, pharmaceutical, and cosmetic products. Lecithin is a product of soy bean oil degumming operations. It is obtained from the hexane extract of flaked soy beans which contains the seed oil and a portion of the phosphatides that are initially present in the soy bean membrane. The exhact is seated with water at 80 °C, and the phosphatides are hydrated rendering them oil insoluble which makes it easy to filler them from the oil. This oil insoluble fraction is termed cmde lecithin and consists of about 70% phosphatides and 30% oil. De-oiled lecithin is obtained by treating the cmde lecithin with acetone. Lecithin is insoluble in acetone and the oil is, and a separation into a 90 to 9r phosphatides fraction can be achieved quite readily. The prior... 

The available methods are suitable only for fractionation according to the basic moieties, whereas phosphatides differing in the lipid moieties are not separated from each other. Compounds which contain differing functional groups in the lipid moiety can be indirectly separated from each other using the following procedure [174] the enzyme phospholipase C from bacteria [Clostridium perfringens or Bacillus cereus) splits off phosphoryl ethanolamine and phosphoryl choline from the phosphatides. The lipophilic hydrolysis products can then be separated as acetyl derivatives (I, II, III) on adsorbent layers. 

A measured volume of a petroleum ether extract of liver containing approximately 6 mg. of phosphatide phosphorus was evaporated just to dryness under reduced pressure in a carbon dioxide atmosphere and the residue was dissolved in methanol. Under these conditions all phosphatides were dissolved by the methanol. This was shown by phosphorus and choline determinations. The methanol solution was made to 100 ml., and 25-ml. aliquots containing approximately 0.060 mg. phosphatide phosphorus per ml. were added to 1.75 g. magnesium oxide in 50-ml. centrifuge tubes. The mixtures were allowed to stand for 25-30 minutes with frequent stirring and then were centrifuged. The methanol was poured off and the magnesium oxide was washed twice with 25-ml. portions of fresh methanol. The combined methanol supernatants were made up to 100 ml. and aliquots were taken for phosphorus and choline determinations. The results are recorded in Table XIII, which shows that the separation by this procedure is just as... 

---