Quantitative determination of phospholipids using the dyes Victoria blue R and B

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.