Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, all of which may well have an impact on the development of atherosclerosis. To investigate the relationship between sphingolipid metabolism and atherosclerosis, we utilized myriocin to inhibit mouse serine palmitoyl-CoA transferase (SPT), the key enzyme for sphingolipid biosynthesis. We injected 8-week-old apoE-deficient mice with myriocin (0.3 mg/ kg/every other day, intraperitoneal) for 60 days. On a chow diet, myriocin treatment caused a significant decrease (50%) in liver SPT activity (p < 0.001), significant decreases in plasma sphingomyelin, ceramide, and sphingosine-1-phosphate levels (54, 32, and 73%, respectively) (p < 0.0001), and a significant increase in plasma phosphatidylcholine levels (91%) (p < 0.0001). Plasma total cholesterol and triglyceride levels demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (42% in root and 36% in en face assays) (p < 0.01). On a high fat diet, myriocin treatment caused marked decreases in plasma sphingomyelin, ceramide, and sphingosine-1-phosphate levels (59, 66, and 81%, respectively) (p < 0.0001), and a marked increase in plasma phosphatidylcholine levels (100%) (p < 0.0001). Total cholesterol and triglyceride demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (39% in root and 37% in en face assays) (p < 0.01). These results indicate that, apart from cholesterol levels, sphingolipids have an effect on atherosclerotic development and that SPT has proatherogenic properties. Thus, inhibition of SPT activity could be an alternative treatment for atherosclerosis.Sphingolipids have many biological functions, including cell membrane formation, signal transduction, and lipid metabolism, and all of these may be related to the development of atherosclerosis. Serine palmitoyl-CoA transferase (SPT) 1 is the rate-limiting enzyme in the biosynthesis of sphingolipids (1). It has long been known that SPT plays an important role in the metabolism of sphingolipids, but its role in other lipid metabolisms and atherosclerosis has not been unequivocally determined. When SPT activity is increased in rat liver (2) and lung (3), sphingolipid formation is likewise increased. The activity of SPT is heightened in the aortas of rabbits fed a high cholesterol diet (4). Two candidate cDNAs for yeast SPT, termed LCB1 and LCB2, have been cloned (5, 6), and the translated sequences indicate that their gene products have a 21% amino acid sequence identity (6). The lack of SPT activity in a yeast strain defective in LCB1 or LCB2, together with the protein similarity data, suggest that the two genes encode subunits of SPT (6). Mouse and human LCB1 and LCB2 cDNA homologues have also been cloned (7,8). In mouse, the two mRNAs have the same tissue distribution (lung, kidney Ͼ brain Ͼ cartilage, skin Ͼ heart Ͼ liver Ͼ muscle), and the ratio of the amounts of the two transcri...

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Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PCspecific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 Mg, and that for PC was 2.5-20 Mg. The mean percentage recovery was 98.0 6 5.2% for SM and 96.6 6 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 6 0.05% for SM and 3.1 6 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.-Hojjati, M. R., and X-C. Jiang. Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine. J. Lipid Res. 2006. 47: 673-676. Supplementary key words phospholipid measurement . large-scale and high-throughput assays . enzymatic measurements Besides cholesterol and triglycerides, plasma also contains phospholipids. Phosphatidylcholine (PC) and sphingomyelin (SM) are two major phospholipids, the former comprising z70% and the latter z20% of the total phospholipids in the circulation (1). In a human case-control study, it was found that both plasma SM and SM/PC ratio are independent risk factors for coronary heart disease (2). It has been known for some time that SM accumulates in atheromas in human and animal models (3, 4). LDL extracted from human atherosclerotic lesions is much richer in SM than LDL from plasma (5, 6). Plasma SM levels in apolipoprotein E knockout mice are four times higher than those in wild-type mice (7), and this may partly explain the increased atherosclerosis in these animals (8). The SM/PC ratio was increased five times in VLDL from hypercholesterolemic rabbits (9). Recently, we and others demonstrated for the first time that administration of myriocin (an inhibitor of SM synthesis) into apolipoprotein E knockout mice dramatically decreased SM, increased PC, thus decreasing the SM/PC ratio in the plasma, and significantly decreased the atherosclerotic lesion area (10, 11). These data suggest that SM might play a promoting role, whereas PC might play a preventive role, in the development of atherosclerosis, and their measurement might provide new insights into atherogenesis in humans and in various mouse models as well. Although the importance of both phospholipids is very obvious, there are no simple, rapid, sensitive, and high-throughput methods for their measurement. Classically, plasma SM and PC were measured by lipid extraction, thin-layer ch...