Protocols for Enzymatic Fluorometric Assays to Quantify Phospholipid Classes

Phospholipids, consisting of a hydrophilic head group and two hydrophobic acyl chains, are essential for the structures of cell membranes, plasma lipoproteins, biliary mixed micelles, pulmonary surfactants, and extracellular vesicles. Beyond their structural roles, phospholipids have important roles in numerous biological processes. Thus, abnormalities in the metabolism and transport of phospholipids are involved in many diseases, including dyslipidemia, atherosclerosis, cholestasis, drug-induced liver injury, neurological diseases, autoimmune diseases, respiratory diseases, myopathies, and cancers. To further clarify the physiological, pathological, and molecular mechanisms and to identify disease biomarkers, we have recently developed enzymatic fluorometric assays for quantifying all major phospholipid classes, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol + cardiolipin, and sphingomyelin. These assays are specific, sensitive, simple, and high-throughput, and will be applicable to cells, intracellular organelles, tissues, fluids, lipoproteins, and extracellular vesicles. In this review, we present the detailed protocols for the enzymatic fluorometric measurements of phospholipid classes in cultured cells.