Protocols for Enzymatic Fluorometric Assays to Quantify Phospholipid Classes

Abstract

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.

Differences in interaction of graphene/graphene oxide with bacterial and mammalian cell membranes

“…Bacterial phospholipids display weak bonding compared to mammalian phospholipids PG and CL, 12 and PC and SM as the major constituents in bacteria and mammalian membranes were selected, respectively 13,14 (ESI Fig. 2 †).…”

“…Based on the abundancy of phospholipids, we selected two phospholipids each as the representatives of the outer leaflet in the plasma membrane of bacteria and mammalian cells. Phosphatidylglycerol (PG) and cardiolipin (CL) are two major constituents in bacteria membranes, 12 while mammalian cell membranes have prominent levels of phosphatidylcholine (PC) and sphingomyelin (SM), 13,14 and thus these four molecules were used in this study.…”

Determination of Phenol with Peroxidase Immobilized on CaCO3

“…One advantage of detection systems is their potential for reusable application. This advantage has been shown in detection systems based on encapsulated enzymes sensor [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ], which reduce analysis costs by reducing enzyme consumption. To determine the feasibility of reusing CaCO 3 –peroxidase particles for phenol concentration detection, we incubated the same CaCO 3 –peroxidase particles five times in different solutions containing 700 ng/mL of phenol, and measured the optical density of each solution.…”

Comparative Lipidomic Analysis of Extracellular Vesicles Derived from Lactobacillus plantarum APsulloc 331261 Living in Green Tea Leaves Using Liquid Chromatography-Mass Spectrometry

“…Given that sphingolipids including Cer participate in vesicle formation and secretion by increasing membrane fluidity and are important regulators for bacterial entry and adherence [54], bacterial EVs possessing different compositions and proportions in sphingolipids according to species may have different potential to affect host cells. Considering the technical advances in lipidomic analysis [30,55], we believe that diverse lipid species will be increasingly detected in bacterial membranes and their extracellular vesicles, although these comprise a minority of the total lipid signal. It is also known that there is a plasticity in the membrane lipid composition of several bacteria under different growth conditions, which was exemplified by CE formation in several members of Actinobacteria and Proteobacteria and cardiolipin and ornithine lipids synthesis in Streptomyces [48,50].…”