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Hydrolysis of platelet-activating factor by purified HDL-associated paraoxonase
Wednesday 12 October 1994: Poster Abstracts HDL and reverse cholesterol transport
bation of this plasma with apo A-l deficient plasma as a source for LCAT forms a-LpA+, In conclusion, our data suggest that pn$Q-LpA-I is formed by free apo A-I in the presence of cells and converted to a-LpA-Ig by an as yet unidentified conversion factor, and subsequently to aLpA-I2 by LCAT. 12021 m,
Effect of lipid monolayer charge on the interaction of CETP with microemulsions Chan RYS, Sawyer WH, Dept. of Biochem., Univ.
of Melbourne, VIC 3052, Australia
The cholesbryl ester transfer protein (CETP) interacts with all lipoprotein classes. Binding of CETP with the lipoprotein surface seems to be the primary event in the neutral lipid transfer reaction. In a model emulsion system, the presence of apolipoproteins A-I and A-II enhanced the cholesteryl ester (CE) transfer. However, the role of surface lipid is unknown. This report concerns the effect of surface charge on the interaction of CETP with the phospholipid monolayer in the absence of apolipoprotein. Microemulsions containing a monolayer of phosphatidylcholine enclosing a core of cholesteryl oleate were prepared by sonication, ultracentrifugation and size exclusion chromatography. The surface charge characteristics of the emulsion particles were altered by incorporating up to 20 mol% of either oleate, phosphatidylserine or stearylamine. The CE transfer activity was measured by a fluorescence assay which measures the excimer fluorescence of cholesteryl I-pyrenedecanoate (Py-CE) in a donor microemulsion as the indicator of CE transfer. The binding of CETP to the emulsion surface was measured from the enhancement of the intrinsic fluorescence of CETP on binding to emulsion particles. Increase in the negative surface charge by the addition of either oleate or phosphatidylserine in the surface monolayer, inhibited the Py-CE transfer promoted by CETP. Under these conditions the binding of CETP to the emulsion particles was also decreased. The inhibition of Py-CE transfer due to phosphatidylserine on the emulsion surface was partially reversed by an increase in the ionic strength of the aqueous phase. On the other hand, stearylamine, which gives a positive charge to the emulsion surface enhanced Py-CE transfer. The results indicate that the activity and the binding of CETP to the surface monolayer of the microemulsion is not favored by negative surface charge. Removal of cholesteryl ester from THP-1 macrophage foam cells using HDL subfractions: the influence of ACAT inhibitor 447C88 and hormonesensitive lipase activity l&&l_&?, Angeli ADR, Yeaman SI*, Wellcome Rex Lab., @
Langley Court, Beckenham. Kent BR3 3B; *Med. Sch., Univ. of Newcastle, Newcastle upon Tyne NE2 4HH, UK
The objective of this study was to determine the effect of HDL and its subfractions on the removal of cholesteryl ester deposits from foam cells. Inhibition of acyl CoA cholesterol acyl transferase (ACAT) and the activity of hormone-sensitive Iipase (HSL) during this process was also investigated. Plasma lipoproteins were prepared by ultracentrifugation using a vertical wall rotor. LDL was acetylated (AcLDL) or oxidized (oxLDL) and HDL subfractions (HDL2, HDLJ) were isolated. THP-1 monocytes differentiated into macrophages using phorbol ester were converted into foam cells by in&b&on wi& AcLDL, OXLDL(50 umglml) or 25-OH cholesterol (2 ., umelmlj ” , in the presence of [*4Cjoleate for 24 h. THP-1 foam cells were then incubated with the ACAT inhibitor 447C88 (I pmM) alone or in the presence of total HDL (HDLT), HDb or HDb for a further 24 h and the amount of cholesteryl [14C]oleate remaining in the foam cells was determined. Incubation of THP-1 macrophages
233
with AcLDL, OXLDLor 25-OH cholesterol led to the formation of cholesteryl ti4C]oleate within the cells. The cholesteryl ester stores could only be partially removed; the most potent subfraction, HDL3, was able to reduce the cholesteryl [‘4C]oleate by 30%. The reduction in cholesteryl [‘4C]oleate was not increased if ACAT was inhibited. HSL activity, responsible for the deesterification of cholesteryl esters, was reduced by up to 50% during foam cell formation. The presence of foam cells within atherosclerotic plaque may be due to the low activity of HSL in foam cells, which reduces the delivery of free cholesterol to HDL. )2041 mMI,
Hydrolysis of platelet-activating factor by purified HDL-associated paraoxonase Abbott CA, Dunington PN, Dept. of Med., Man-
chester Royal Infirmary, Manchester Ml3 9WL+ UK
Human serum paraoxonase is an enzyme which is associated with a specific HDL particle containing apolipoproteins Al and J. We have previously shown that paraoxonase purified from human HDL inhibited lipid peroxidation when LDL was incubated under oxidizing conditions. Platelet-activating factor (PAF) is an ether phospholipid which is involved in the mediation of inflammatory responses in the artery wall. It is hydrolyzed by PAF acetyl hydrolase (PAFAH), which is active on both HDL and LDL and which is also capable of hydrolyzing oxidized phospholipids. HDL-associated PAFAH might be the same enzyme as paraoxonase. Paraoxonase was therefore purified to homogeneity from human HDL using a combination of affinity, anion-exchange and hydrophobic interaction chromatography. A single protein band was found on both SDS-PAGE and isoelectric focusing which reacted with a monoclonal antibody against human paraoxonase on Western blotting. Paraoxonase hydrolysed PAF at a rate of 10 nmol/minlmg with a Km of 10.1 PM but its activity was approximately 1000 times lower than that of LDL-associated PAFAH. Our results thus indicate that paraoxonase can contribute to PAFAH activity on HDL but that it is not the same enzyme. Both enzymes may act in concert to hydrolyse lipid peroxides generated on LDL. in the activity of reverse cholesterol 12051Alterations transport: a contributing factor to the hyperlipidemia of nepbrotic syndrome? Wood GM, Bhatnagar D, Mackness MI, Haynes B, Durrington PN, Short CD, Depts. of Med. and Neohroloav, Univ. of Manchester, Manchester i?oyai Inj?mary, 9WL, UK
O*xford ;;id, Manchester Ml3
The dyslipidemia associated with proteinuria is likely to increase coronary risk. It has now become evident that transfer of cholesterol back to the liver, the reverse cholesterol pathway, is an important consideration in the development of atherosclerosis. Cholesteryl esters (CE) generated by LCAT are transferred from HDL to LDL or to VLDL by the action of cholesteryl ester transfer protein (CETP). LCAT and CET activities were measured in 18 non-diabetic nephrotic patients with a mean urinary albumin excretion of 10.8 g/day, mean creatinine clearance of 50.6 ml/min and a mean serum albumin of 32.7 g/l. LCAT activity was determined by a modification of the Stokke and Norum method. An isotopic assay, employing endogenous lipoproteins, was used to determine rate of transfer of cholestervl esters from HDL to LDLNLDL. The following values (me& rt SD) are compared with 27 healthy controls: TC 8.2 +2 vs 5.9 kO.9 (P
Atherosclerosis X, Montreal, October 1994
bation of this plasma with apo A-l deficient plasma as a source for LCAT forms a-LpA+, In conclusion, our data suggest that pn$Q-LpA-I is formed by free apo A-I in the presence of cells and converted to a-LpA-Ig by an as yet unidentified conversion factor, and subsequently to aLpA-I2 by LCAT. 12021 m,
Effect of lipid monolayer charge on the interaction of CETP with microemulsions Chan RYS, Sawyer WH, Dept. of Biochem., Univ.
of Melbourne, VIC 3052, Australia
The cholesbryl ester transfer protein (CETP) interacts with all lipoprotein classes. Binding of CETP with the lipoprotein surface seems to be the primary event in the neutral lipid transfer reaction. In a model emulsion system, the presence of apolipoproteins A-I and A-II enhanced the cholesteryl ester (CE) transfer. However, the role of surface lipid is unknown. This report concerns the effect of surface charge on the interaction of CETP with the phospholipid monolayer in the absence of apolipoprotein. Microemulsions containing a monolayer of phosphatidylcholine enclosing a core of cholesteryl oleate were prepared by sonication, ultracentrifugation and size exclusion chromatography. The surface charge characteristics of the emulsion particles were altered by incorporating up to 20 mol% of either oleate, phosphatidylserine or stearylamine. The CE transfer activity was measured by a fluorescence assay which measures the excimer fluorescence of cholesteryl I-pyrenedecanoate (Py-CE) in a donor microemulsion as the indicator of CE transfer. The binding of CETP to the emulsion surface was measured from the enhancement of the intrinsic fluorescence of CETP on binding to emulsion particles. Increase in the negative surface charge by the addition of either oleate or phosphatidylserine in the surface monolayer, inhibited the Py-CE transfer promoted by CETP. Under these conditions the binding of CETP to the emulsion particles was also decreased. The inhibition of Py-CE transfer due to phosphatidylserine on the emulsion surface was partially reversed by an increase in the ionic strength of the aqueous phase. On the other hand, stearylamine, which gives a positive charge to the emulsion surface enhanced Py-CE transfer. The results indicate that the activity and the binding of CETP to the surface monolayer of the microemulsion is not favored by negative surface charge. Removal of cholesteryl ester from THP-1 macrophage foam cells using HDL subfractions: the influence of ACAT inhibitor 447C88 and hormonesensitive lipase activity l&&l_&?, Angeli ADR, Yeaman SI*, Wellcome Rex Lab., @
Langley Court, Beckenham. Kent BR3 3B; *Med. Sch., Univ. of Newcastle, Newcastle upon Tyne NE2 4HH, UK
The objective of this study was to determine the effect of HDL and its subfractions on the removal of cholesteryl ester deposits from foam cells. Inhibition of acyl CoA cholesterol acyl transferase (ACAT) and the activity of hormone-sensitive Iipase (HSL) during this process was also investigated. Plasma lipoproteins were prepared by ultracentrifugation using a vertical wall rotor. LDL was acetylated (AcLDL) or oxidized (oxLDL) and HDL subfractions (HDL2, HDLJ) were isolated. THP-1 monocytes differentiated into macrophages using phorbol ester were converted into foam cells by in&b&on wi& AcLDL, OXLDL(50 umglml) or 25-OH cholesterol (2 ., umelmlj ” , in the presence of [*4Cjoleate for 24 h. THP-1 foam cells were then incubated with the ACAT inhibitor 447C88 (I pmM) alone or in the presence of total HDL (HDLT), HDb or HDb for a further 24 h and the amount of cholesteryl [14C]oleate remaining in the foam cells was determined. Incubation of THP-1 macrophages
233
with AcLDL, OXLDLor 25-OH cholesterol led to the formation of cholesteryl ti4C]oleate within the cells. The cholesteryl ester stores could only be partially removed; the most potent subfraction, HDL3, was able to reduce the cholesteryl [‘4C]oleate by 30%. The reduction in cholesteryl [‘4C]oleate was not increased if ACAT was inhibited. HSL activity, responsible for the deesterification of cholesteryl esters, was reduced by up to 50% during foam cell formation. The presence of foam cells within atherosclerotic plaque may be due to the low activity of HSL in foam cells, which reduces the delivery of free cholesterol to HDL. )2041 mMI,
Hydrolysis of platelet-activating factor by purified HDL-associated paraoxonase Abbott CA, Dunington PN, Dept. of Med., Man-
chester Royal Infirmary, Manchester Ml3 9WL+ UK
Human serum paraoxonase is an enzyme which is associated with a specific HDL particle containing apolipoproteins Al and J. We have previously shown that paraoxonase purified from human HDL inhibited lipid peroxidation when LDL was incubated under oxidizing conditions. Platelet-activating factor (PAF) is an ether phospholipid which is involved in the mediation of inflammatory responses in the artery wall. It is hydrolyzed by PAF acetyl hydrolase (PAFAH), which is active on both HDL and LDL and which is also capable of hydrolyzing oxidized phospholipids. HDL-associated PAFAH might be the same enzyme as paraoxonase. Paraoxonase was therefore purified to homogeneity from human HDL using a combination of affinity, anion-exchange and hydrophobic interaction chromatography. A single protein band was found on both SDS-PAGE and isoelectric focusing which reacted with a monoclonal antibody against human paraoxonase on Western blotting. Paraoxonase hydrolysed PAF at a rate of 10 nmol/minlmg with a Km of 10.1 PM but its activity was approximately 1000 times lower than that of LDL-associated PAFAH. Our results thus indicate that paraoxonase can contribute to PAFAH activity on HDL but that it is not the same enzyme. Both enzymes may act in concert to hydrolyse lipid peroxides generated on LDL. in the activity of reverse cholesterol 12051Alterations transport: a contributing factor to the hyperlipidemia of nepbrotic syndrome? Wood GM, Bhatnagar D, Mackness MI, Haynes B, Durrington PN, Short CD, Depts. of Med. and Neohroloav, Univ. of Manchester, Manchester i?oyai Inj?mary, 9WL, UK
O*xford ;;id, Manchester Ml3
The dyslipidemia associated with proteinuria is likely to increase coronary risk. It has now become evident that transfer of cholesterol back to the liver, the reverse cholesterol pathway, is an important consideration in the development of atherosclerosis. Cholesteryl esters (CE) generated by LCAT are transferred from HDL to LDL or to VLDL by the action of cholesteryl ester transfer protein (CETP). LCAT and CET activities were measured in 18 non-diabetic nephrotic patients with a mean urinary albumin excretion of 10.8 g/day, mean creatinine clearance of 50.6 ml/min and a mean serum albumin of 32.7 g/l. LCAT activity was determined by a modification of the Stokke and Norum method. An isotopic assay, employing endogenous lipoproteins, was used to determine rate of transfer of cholestervl esters from HDL to LDLNLDL. The following values (me& rt SD) are compared with 27 healthy controls: TC 8.2 +2 vs 5.9 kO.9 (P
Atherosclerosis X, Montreal, October 1994