Metabolism and intracellular distribution of a fluorescent analogue of phosphatidic acid in cultured fibroblasts.

We have shown that a fluorescent compound, C6-NBD-PA, behaves as an analogue for phosphatidic acid, an important intermediate in glycerolipid biosynthesis. This derivative is preferentially transferred from phospholipid vesicles to cultured Chinese hamster fibroblasts at 2 degrees C, while the C6-NBD-PA-derived fluorescence is localized at the nuclear membrane, endoplasmic reticulum, and mitochondria. Extraction and analysis of the fluorescent lipids associated with the cells after treatment with vesicles at 2 degrees C or 37 degrees C revealed that a large fraction of the fluorescent phosphatidic acid is converted to fluorescent diglyceride, phosphatidylcholine, and triglyceride. Although we do not yet know how accurately the metabolism and intracellular distribution of C6-NBD-PA and its metabolites reflect those of endogenous phosphatidic acid, it is encouraging that this fluorescent analogue is apparently metabolized through the diglyceride pathway to give fluorescent analogues of diglyceride, triglyceride, and phosphatidylcholine. This metabolism suggests that the presence of the fluorescent group on the acyl chain of the phosphatidic acid analogue does not inhibit the enzymes involved in phosphatidic acid metabolism. We conclude that fluorescent lipid analogues such as C6-NBD-PA may be useful in correlating biochemical studies of lipid metabolism with studies of the intracellular localization of lipid metabolites by fluorescence microscopy.