Enzymatic determination of phosphatidylcholine, sphingomyelin and phosphatidylglycerol in lipid dispersions, blood cell membranes and rat pulmonary surfactant.
A new micromethod for the determination of sphingomyelin in samples suspended in aqueous solutions, and modified micromethods for determining phosphatidylcholine and phosphatidylglycerol were used to determine phosphatidylcholine and sphingomyelin (detection limits of 1.8 mumol/l), and phosphatidylglycerol (detection limit of 2.3 mumol/l) in lipid dispersions, membranes from sheep erythrocytes and platelets, and pulmonary surfactants from rats of different ages and rats maintained under normobaric hyperoxia for 2 days prior to their sacrifice. The procedures are easy to perform, accurate, require less sample than conventional methods and can also be applied directly to aqueous samples. Phospholipase C and sphingomyelinase were used to release phosphorylcholine from phosphatidylglycerol and sphingomyelin, respectively. The choline released from phosphorylcholine by alkaline phosphatase is reconverted to phosphorylcholine by ATP and choline kinase. In the phophatidylglycerol determination, phospholipase D was used to release glycerol and phosphatidate. The glycerol formed was converted to glycerolphosphate using ATP and glycerol kinase. In all cases, the ADP thus formed was determined by following the enzymatic conversion of NADH to NAD at 340 nm in an coupled pyruvate kinase/lactate dehydrogenase system. Significant variations in the phospholipid composition of rat pulmonary surfactant were found during development; in particular there was an increase in the phosphatidylglycerol content of adult rats as compared with younger rats. Hyperoxia produced changes in the phosphatidylglycerol content of surfactant from adult rats, but not from 2-day old rats.
Encinar
J A
JA
Departamento de Bioquímica y Biología Molecular, Universidad de Salamanca, Spain.
Ludeña
M D
MD
Sánchez-Yagüe
J
J
Llanillo
M
M
D000818:Animals; D001681:Biological Assay / Q000379:methods; D001792:Blood Platelets / Q000737:chemistry; D004910:Erythrocyte Membrane / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000032:analysis*; D010715:Phosphatidylglycerols / Q000032:analysis*; D010767:Phosphorylcholine / Q000378:metabolism; D011663:Pulmonary Surfactants / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D012756:Sheep; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013109:Sphingomyelins / Q000032:analysis*; D010738:Type C Phospholipases / Q000378:metabolism
D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D011663:Pulmonary Surfactants; D013109:Sphingomyelins; D010767:Phosphorylcholine; D010738:Type C Phospholipases; D013108:Sphingomyelin Phosphodiesterase