An original method for rapid serial determination of phospholipid biosynthesis. Applications to mammalian lymphocytic cells and a lower eucaryote, Plasmodium falciparum.
A rapid, convenient, and efficient method is presented to measure phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol biosynthesis from [3H]choline, [3H]ethanolamine, and [3H]inositol, respectively. After incubation of the cells in 96-multi-well dishes with the appropriate radioactive precursor, cells were lysed with water and the water-insoluble materials, particularly cellular membranes which contain the bulk of phospholipids, were serially collected on glass-fiber papers using a cell harvester. The method was first applied to human lymphocytic cell lines then adapted to Plasmodium falciparum-infected human erythrocytes which in both cases allowed recovery of more than 90% of the newly biosynthesized phospholipids. With this quick method, adapted to short incubation periods (less than 5 h), we were able to determine optimal conditions such as the best medium (RPMI devoid of serum, thus avoiding interference from endogenous precursors, notably choline present in significant quantities in serum) and the lowest specific activity to be used for each radioactive precursor and the minimum quantity of cells. This method could be adapted to other cell systems, provided that the precursors are specific to phospholipids and that the bulk of biosynthesized phospholipids are present as membrane components. Finally, by this method the activity of effectors of phospholipid metabolism can be tested on a large scale, thus allowing rapid screening of original molecules specifically affecting cellular phospholipid metabolism.
Ancelin
M L
ML
CNRS U.A. 530, Département Biologie-Santé, Montpellier, France.
Vialettes
F
F
Vial
H J
HJ
D000605:Amino Alcohols / Q000494:pharmacology; D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy; D009101:Multiple Myeloma / Q000378:metabolism; D010712:Phosphatidic Acids / Q000032:analysis*; D010713:Phosphatidylcholines / Q000032:analysis; D010714:Phosphatidylethanolamines / Q000032:analysis; D010716:Phosphatidylinositols / Q000032:analysis; D010743:Phospholipids / Q000096:biosynthesis*; D010963:Plasmodium falciparum / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000605:Amino Alcohols; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010743:Phospholipids; D000644:Quaternary Ammonium Compounds; C013912:decyltrimethylammonium; C006506:2-amino-1-butanol