4.P.362 Lecithin: Cholesterol acyltransferase (LCAT) knockout mice: A new animal model for human LCAT-deficiency

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Thursday 9 October 1997: Posters HDL/reverse cholesterol transport

4 .P.358 Cholesteryl ester transfer rate from HDL to triacylglycerol-rich emulsions depends on the triacylglycerol fatty add chain length P.M . Cazita, V .S . Nunes, L.N. Castilho, E .C .R. QuintAo . Lipids Lab, LIM 10, University of Sao Paulo Medical School, Sao Paulo, SP Brazil The aim of this study was to evaluate the influence of saturated (SAT) and monounsaturated (MONO) triacylglycerol (TG) emulsions (EM) that differ according to their fatty acid chain length on the transfer rate of 14C-cholesteryl oleate (1dC-CO) from HDL to EM . EM were prepared by sonication of a lipid mixture (2% cholesterol, 6% cholesteryl oleate, 23% lecithin and 69% TG), where the composition of TG varied according both to the degree fatty acid saturation and chain length as shown below : SAT: 50% Cl 1 : 0 + 50% C18 : 1 ; 50% C12 : 0 + 50% C18 : 1 ; 50% C14: 0 + 50% Cl8 : 1 ; 50% C16 : 0 + 50% C18 : 1 ; MONO : 50% Cl 1 : 1 + 50% C18 : 1 ; 50% C12 :1 + 50% C18 : 1 ; 50% C16 :1 + 50% C18 : 1 ; 100% C18: 1 . HDL was obtained from a healthy donor plasma pool, labeled with 14C-CO (120 sg CO), and incubated with the EM (500 .tg de TG) and plasma with DTNB (D > 1 .21 g/mL) as the CETP source at 37°C for 4 h . HDL was separated from EM by discontinues density gradient ultracentrifugation for 30 min at 4°C and 190 .000 g . 14C-CO transfer from HDL to EM was determined by the measurement of radioactivity in the EM fraction . Data comparing rates of transfer by the Mann-Whitney test between different EM were corrected for the blank values and were expressed per smol TG : a : Cl 1 :0 C14 :0 and C16:0, C12:0#C14 :0 and C16 :0 ; b : C11 :1y C16 :1 and C18 :1, C12:1#C16 :1 and C18 :1 . Median of % transfer/µmot TG 10.7 SAT Cl 1 :0(n=15) 13 .1 MONO CI I :I(n=12) 10.6 C12 :0(n=21) 12 .3 C12:1 (n = 12) C16:I(n=12) 20 .4b C14 :0(n=23) 17.2a C16 :0(n=21) 16.4a C18:1(n=12) 20 .7b (a) p < 0 .001 ; (b) p < 0 .0001 . We conclude that CETP-mediated transfer rates of 14C-CO from HDL to EM increase according to the EM-TG fatty acid chain length and not to the degree of saturation. 4 .P.359 Cholesteryl ester-HDL transfer rate to apoB-containing lipoproteins is distinctly influenced by glycation of the HDL components and of CETP M . Passarellil, S . Catanozil, E .R. Nakandakaret, J .C . Rocha', A .F.M. Shimabukurot, R .E . Morton, E .C.R . Quintaot . /Lipids Lab. Univ. Sao Paulo Medical School (LIM 10), Sao Paulo, Brazil; 'Dept . of Cell Biology, Cleveland Clinic Foundation, Clinic Foundation, Ohio, USA Alterations in the reverse cholesterol transport (RCT) system ascribed to glycation of lipoproteins (LP) have been considered critical in the process of atherogenesis in diabetes mellitus (DM) [Passarelli et al 1995 Atherosclerosis 115 (suppl .) : S102] . The transfer rate of cholesteryl ester (CE) from HDL to apoB-containing LP was investigated in order to measure the influence of glycation of the cholesteryl ester transfer protein (CETP) and of lipid or protein HDL components . HDL was delipidated with an ethanol :ethyl ether mixture (3 :2, v :v) and 0 .02 mM BHT. The lipid phase was radiolabeled with cholesteryl oleate [Cholesteryl-4-14C] . Partially purified CETP, HDL lipid and protein were independently glycated on a 200 mM glucose solution . HDL was next reconstituted by ultrasonication and repurified . Plasma D > 1 .21 g/mL with DTNB was utilized as the source of CETP on CE transfer assays carried out at 37°C and at 4°C . Data as median express % transfer of CE from HDL to LDL or VLDL, respectively during 4 or 3 h periods . C = control ; G = glycated . Reconstituted HDL (rec) experimental groups were : LG = lipid was G ; PG = protein was G ; LPG = lipid and protein fractions were G . Highly purified CETP C elicited 23% transfer from 14C-CE-HDL to native LDL as compared to 3 .3% transfer with CETP G . during 4 h (p < 0 .0001). Glycation of the protein but not of the lipid fraction of HDL enhances the transfer rate of 14C-CE from HDL to apoB-containing LP, whereas glycation of the CETP markedly diminishes its CE transfer activity . These results suggest potential alterations on the RCT system in DM . rec C rec LG rec PG rec LPG LDL C 72 .5 71 .5 76 .51 b 73 .4' 71 .8' 70.0 75 .51 b 69.0 LDLG VLDL C 40.0 38 .0 47 .0b•c 37.0 VLDL G 38 .5 36.5 47 .5b•c 39.0 Mann-Whitney test (n = 10) : 1 p < 0 .0005 vs. rec C; b p < 0.0001 vs . rec LG and rec LPG ; c p < 0 .05 vs. rev C ;' p < 0 .05 vs . recLG and LPG ; e p < 0 .05 vs. recLG.

4 .P.360 Large unilamellar vesicles mediate cholesterol mobilization and regression of atherosclerotic lesions in cholesterol-fed rabbits W.V. Rodrigueza, S .K. Klimuk, P.H. Pritchard, M.J . Hope . Allegheny University of the Health Sciences, Philadelphia, PA, USA ; University of British Columbia, Vancouver, BC, Canada Cholesterol-free phospholipid liposomes have been proposed as synthetic mediators of reverse cholesterol transport in vivo . We now tested the ability of large unilamellar phospholipid vesicles (LUVs ; diameter = 100 nm diameter) to shrink lesions in cholesterol-fed rabbits . Forty eight rabbits were divided into two diet groups fed standard rabbit chow or fed a 0 .5% cholesterolenriched diet to induce the formation of atherosclerotic lesions . Prior to the initiation of LUV therapy, the cholesterol diet was ceased and all animals were returned to standard rabbit chow . The treatment protocol consisted of a total of 10 bolus injections of vesicles, at a phospholipid dose of 300 mg/kg body weight or the equivalent volume of saline, with one injection given to each animal every 10 days . LUV injections brought about a large movement of cholesterol into the blood pool and resulted in a significant reduction in the cholesterol content as well as the degree of surface plaque involvement of aortic tissue in atherosclerotic animals . Most notably, the thoracic aorta of LUV-treated animals exhibited a 48% reduction in tissue cholesterol content per gram of protein compared to saline-treated controls . Histochemical analyses revealed that aortas from animals receiving repeated injections of LUVs displayed less cholesterol deposists in lesions, and a moderate reduction in intimal-to-medial thickness . This depletion of atheroma cholesterol induced by LUV therapy was observed even though animals possessed persistent elevated plasma cholesterol levels after the cholesterol-enriched diet was ceased . These results suggest that repeated injections of LUVs may be a useful therapy in the management of atherosclerosis . 4.P.361

Lack of an effect of physical exercise on the serum concentration of HDL subfractions containing ape A-I with and without apoA-II A. Rovellini, A. Branchi, L . Arcangeli 1, L. Re', L. Marchetti', D. Sommariva2 . Institute of Internal Medicine and Medical Physiopathology, University of Milan, Ospedale Maggiore IRCCS ; 'Laboratory of Clinical Analysis; 2Department of Internal Medicine, Hospital of Bollate, Bollate, Italy In sedentary individuals, sufficient amounts of exercise training have been reported to induce an increase of HDL cholesterol level . We studied whether a single bout of exercise in non trained subjects may elicit changes in HDL and its subfractions . The serum level of HDL cholesterol and of HDL particles, defined by the presence of apolipoprotein A-I (LpA-I) or of both apolipoprotein A-I and A-II (LpA-I :A-II), was measured in 31 subjects before and 10 minutes after 30 minutes exercise on a calibrated stationary cycle ergometer . The exercise protocol consisted in 75-175 W of maximum work ; the average 02 uptake was 1 .684 I/min (85% VO2 max) . After exercise, HDL cholesterol increased by 17%, LpA-I by 11% and LpA-I :A-II by 12% . The statistical significance of the changes was lost when post exercise data were adjusted for the estimated alterations in plasma volume . Before After P HDL cholesterol 46.1 t 1 .46 54.0 t 1 .71