Evidence for lack of phosphatidic acid and phospholipase D activity in milk

Phosphatidic acid could not be demonstrated when milk was assayed for phospholipase D activity even when lecithin was added. The concentration of "free" choline in milk remained the same before and after addition of lecithin. However, phosphatidic acid and an increased concentration of "free" choline were demonstrated when cabbage phospholipase D was added to raw milk. The concentration of phosphatidic acid and "free" choline in milk increased appreciably when both lecithin and cabbage phospholipase D were added to raw milk. Phospholipase D activity in milk was assayed at 23.5 C, pH 5.6, in the presence of 10% vol/vol of diethyl ether and .015M calcium chloride. "Free" choline was determined by adjusting the acid whey from milk to pH 11 and adding a freshly prepared 3% methanolic solution of ammonium reineckate. The precipitated choline reineckate was washed, dissolved in acetone, and its absorbance measured at 520 nm. The absence or presence of phosphatidic acid was shown by thin-layer chromatography. These results refute a previous report which proposed that both phosphatidic acid and phospholipase D were in milk.