Lipoprotein Sphingomyelin Research Articles

Application of enzymatic fluorometric assays to quantify phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in human plasma lipoproteins

Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM) are important surface components of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL). However, the pathophysiological roles of PC, PE and SM in lipoproteins have not been well characterized owing to the difficulties in quantifying phospholipid classes in lipoproteins. In this study, we assessed the precision and accuracy of the enzymatic fluorometric assays for measuring PC, PE and SM in VLDL, LDL and HDL, which were isolated from human plasma by ultracentrifugation. The within-run coefficients of variation (CV) for the measurements of PC, PE and SM in lipoproteins were 1.5–2.8 %, 1.1–2.4 % and 0.9–2.3 %, respectively, whereas the between-run CVs for the PC, PE and SM assays were 2.7–4.7 %, 2.1–4.5 % and 1.6–3.3 %, respectively. Excellent linearity and almost complete recovery were achieved for all assays measuring PC, PE and SM in VLDL, LDL and HDL. Our preliminary results using these enzymatic fluorometric assays suggested that the phospholipid compositions were different among VLDL, LDL and HDL. In conclusion, we established high-throughput enzymatic fluorometric assays to quantify PC, PE and SM in human plasma VLDL, LDL and HDL, which will be useful for further investigation of pathophysiological roles of phospholipids in lipoproteins.

Inhibition of endothelial lipase activity by sphingomyelin in the lipoproteins.

Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.

Adeno‐associated virus‐mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E−/− mice

The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis. Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma. Plasma sphingomyelin levels were reduced at early but not later time points after the administration of AAV8-ASM despite persistently elevated circulating ASM. No change in serum lipoprotein levels was observed. Thirteen or 17 weeks after the administration of AAV8-ASM, the amount of plaque formation in the aortic sinus was comparable to that of mice treated with a control AAV. Unexpectedly, the lesion area of the entire aorta was reduced significantly in the AAV8-ASM virus-treated group. Hepatic expression and secretion of ASM into the circulation did not accelerate or exacerbate, but rather decreased, lesion formation in ApoE(-/-) mice. Thus, plasma ASM activity does not appear to be rate limiting for plaque formation during atherogenesis.

Long-term ethanol consumption impairs reverse cholesterol transport function of high-density lipoproteins by depleting high-density lipoprotein sphingomyelin both in rats and in humans

Moderate alcohol consumption has been linked to lower incidence of coronary artery disease due to increased plasma high-density lipoprotein (HDL), whereas heavy drinking has the opposite effect. Because of the crucial role of HDL in reverse cholesterol transport and positive correlation of HDL sphingomyelin (SM) content with cholesterol efflux, we have compared HDL SM content with its reverse cholesterol transport capacity both in rats fed ethanol on long-term basis and alcoholic individuals. In rats, SM HDL content was decreased in the ethanol group (−15.4%, P < .01) with a concomitant efflux decrease (−21.0%, P < .01) compared to that in controls. Similarly, HDL from the ethanol group, when compared with HDL from the control group, exhibited 13.8% ( P < .05) less cholesterol uptake with control-group hepatocytes and 35.0% ( P < .05) less cholesterol uptake with ethanol-group hepatocytes. Conversely, hepatocytes from the ethanol group, when compared with hepatocytes from the control group, exhibited 31.0% ( P < .01) less cholesterol uptake with control-group HDL and 48.0% ( P < .01) less with ethanol-group HDL. In humans, SM content in plasma HDL was also decreased in chronically alcoholic individuals without liver disease (−51.5%, P < .01) and in chronically alcoholic individuals with liver disease (−51.3%, P < .01), compared with nondrinkers. Concomitantly, in alcoholic individuals without liver disease, both efflux and uptake were decreased by 83.0% and 54.0% ( P < .01), respectively, and in chronically alcoholic individuals with liver disease by 84.0% and 61.0% ( P < .01), respectively, compared with nondrinkers. Based on these findings, we conclude that long-term ethanol consumption significantly impairs not only cholesterol efflux function of HDL by decreasing its SM content but also cholesterol uptake by affecting presumably hepatocyte receptors for HDL.

Modulation of lipoprotein metabolism by inhibition of sphingomyelin synthesis in ApoE knockout mice

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.

Regulation of the selective uptake of cholesteryl esters from high density lipoproteins by sphingomyelin

Although sphingomyelin (SM) is a major phospholipid in lipoproteins as well as in the membrane rafts where the scavenger receptor class B type I (SR-BI) is localized, its possible role in the selective uptake of cholesteryl ester (CE) by the SR-BI-mediated pathway is unknown. We investigated the effect of SM in lipoproteins and cell membranes on the selective uptake in three different cell lines: SR-BI-transfected CHO cells, hepatocytes (HepG2), and adrenocortical cells (Y1BS1). Incorporation of SM into recombinant high density lipoprotein (rHDL) containing labeled CE resulted in up to 50% inhibition of the selective uptake of CE in all three cell lines. This inhibition was completely reversed by treatment of rHDL with sphingomyelinase (SMase). Selective uptake from plasma HDL was activated by 22-72% after treatment of HDL with SMase. In addition, pretreatment of the cells with SMase resulted in stimulation of CE uptake from rHDL by CHO and Y1BS1, although not by HepG2. Incorporation of ceramide into rHDL resulted in up to 2-fold stimulation of CE uptake, although pretreatment of cells with egg ceramide had no significant effect. These results show that SM and ceramide in the lipoproteins and the cell membranes regulate the SR-BI-mediated selective uptake of CE, possibly by interacting with the sterol ring or with SR-BI itself.

The change of composition as well as the concentration of serum Lp(a) can be a sign for end-stage renal failure

Plasma sphingomyelin (SM) has been suggested as a risk factor for coronary heart disease independent of cholesterol levels. A decrease of SM in lipoproteins is known to improve the activities of lecithin:cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) in vitro. Inhibition of SM biosynthesis may reduce lipoprotein SM content and thus improve cholesterol distribution in lipoproteins by enhancing reverse cholesterol transport and clearance of triglyceride-rich lipoproteins. To examine this hypothesis, ApoE KO mice were fed a western diet and treated for 4 weeks with various concentrations of myriocin, a specific inhibitor of serine palmitoyltransferase. Myriocin treatment lowered plasma cholesterol and TG levels in a dose-dependent manner. In addition, myriocin treatment reduced cholesterol contents in VLDL and LDL and elevated HDL-cholesterol. Observed lipid-lowering effects of myriocin were associated with suppression of HMG CoA reductase and fatty acid synthase via reduced levels of SREBP-1 RNA and protein. Induction of apoAI and lecithin:cholesterol acytransferase (LCAT) in the liver by myriocin was associated with an increased HDL. Lesion area and macrophage area were also diminished in the cuffed femoral artery of ApoE KO mice. In conclusion, inhibition of sphingolipid biosynthesis can be a novel therapeutic target for dyslipidemia and atherosclerosis.

Oxidation of cholesterol in low density and high density lipoproteins by cholesterol oxidase.

The cholesterol oxidase-catalyzed oxidation of cholesterol in native low density (LDL) and high density lipoproteins (HDL3) as well as in monolayers prepared from surface lipids of these particles, has been examined. The objective of the study was to compare the oxidizability of cholesterol, and to examine the effects of lipid packing on oxidation rates. When [3H]cholesterol-labeled lipoproteins were exposed to cholesterol oxidase (Streptomyces sp.), it was observed that LDL [3H]cholesterol was oxidized much faster than HDL3 [3H]cholesterol. This was true both at equal cholesterol concentration per enzyme unit, and at equal amounts of lipoprotein particles per enzyme unit. About 95% of lipoprotein [3H]cholesterol was available for oxidation. The complete degradation of lipoprotein sphingomyelin by sphingomyelinase (Staphylococcus aureus) resulted in a 10-fold increase in the rate of LDL [3H]cholesterol oxidation, whereas the effects on rates of HDL3 [3H]cholesterol oxidation were less dramatic. A monolayer study with LDL surface lipids indicated that degradation of sphingomyelin loosened the lipid packing, because the ceramide formed occupied a smaller surface area than the parent sphingomyelin, and since the condensing effect of cholesterol on sphingomyelin packing was lost. The effects of sphingomyelin degradation on lipid packing in monolayers of HDL3-derived surface lipids were difficult to determine from monolayer experiments. Based on the finding that cholesterol oxidases are surface pressure-sensitive with regard to their catalytic activity, these were used to estimate the surface pressure of intact LDL and HDL3. The cut-off surface pressure of a Brevibacterium enzyme was 25 mN/m and 20 mN/m in monolayers of LDL and HDL3-derived surface lipids, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake and utilization of double-labeled high-density lipoprotein sphingomyelin in isolated brain capillaries of adult rats.

Isolated rat brain capillaries were incubated in the presence of high-density lipoprotein (HDL) containing [stearic acid-14C, (methyl-3H)choline]sphingomyelin. This double-labeled sphingomyelin was taken up in a concentration-dependent manner. Cerebral capillary-associated sphingomyelin had a 3H/14C ratio close to that of the incubation medium, a result indicating uptake of sphingomyelin without prior hydrolysis. TLC of lipid extracted from capillaries showed that part of the sphingomyelin (up to 40%) was hydrolyzed in the brain capillaries to ceramide and free fatty acids. The hydrolysis was proportional to the amount of incorporated sphingomyelin and reached a plateau when the HDL sphingomyelin concentration in the medium was 237 nmol/ml. The results of "pulse-chase" experiments showed that the choline moiety of sphingomyelin was recovered in the incubation medium after the chase period and that there was no redistribution of liberated choline in phosphatidyl-choline of capillaries.

Turnover and uptake of double-labelled high-density lipoprotein sphingomyelin in the adult rat

Rat HDL containing [ stearic acid- 14C, ( methyl- 3H) choline]sphingomyelin was prepared by incubating labelled sphingomyelin liposomes with serum. HDL was then separated by ultracentrifugation and purified by gel-filtration chromatography. The maximum transfer was reached when 1.5 μmol sphingomyelin was incubated in the presence of 1 ml of serum at 37° C for 1 h. When transfer was limited to a 5–7% increase in HDL mass, no significant change was observed in the HDL electrophoretic pattern, and rats could therefore be injected with this type of HDL under physiological conditions. Plasma radioactivity decay was followed for 24 h, and the recovery of both isotopes in 11 tissues was studied 24 h after the injection. The decay in plasma of both isotopes followed three exponential phases. During the first two phases, both isotopes disappeared with the same velocity ( t 1 2 = 12.8 and 98–105 min for the first and second phases, respectively). 10 h after injection, 3H had disappeared more slowly than 14C ( t 1 2 = 862 and 502 min for 3H and 14C, respectively) and 24 h after injection, only 1.5% of 14C and 2.5% of 3H remained in the plasma. This radioactivity was located mainly in HDL (80–85% for 3H and 14C, with a 3H/ 14C ratio close to that of injected sphingomyelin, and in VLDL, with the same isotopic ratio as that of liver lipids. Some 3H was associated with non-lipoprotein proteins. 17.5% of 3H and 23.4% of 14C were recovered in the liver, 1.6% of each isotope in erythrocytes, and 1.4% of 3H and 0.6% of 14C in kidney. Less than 1% of each isotope was recovered in each of the other tissues. Phosphatidylcholine was the lipid most labelled, and in several tissues sphingomyelin had a 3H/ 14C ratio close to that of injected sphingomyelin, showing an uptake without prior hydrolysis.