Inhibition of lecithin: Cholesterol acyltransferase by cholesterol oxides

Wednesday June 28,200O: Poster Abstracts P: W22 Reverse Cholesterol Transport

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(PKD) in 2 cases of 1 family; (probable) angina pectoris in 2 cases (ages of 63 and 77); SAH in 1 case of PKD family; liver cirrhosis in 1 case; COPD in 1 case; FH in 1 case; and DM in 1 case. Conclusions: Prevalence of premature atherosclerotic diseases was not increased in homozygous CETP deficiency despite hypercholesterolemia.

IWeP30

W22

Inhibition of lecithin: Cholesterol acyltransferase cholesterol oxides

by

EC. Pincinato’, A. Sevaniru?, D.S.P. Abdalla’. ‘Depto. of Clin and Toxicol. Analyses, FCF USP Scio Paulo, SP Brazil; Inst. Toxicol., University of Southern California, IA, CA, USA Objective: To investigate the effect of cholesterol oxides on esteritication of cholesterol by LCAT. Methods: HDL particles enriched with increasing concentration of cholesterol oxides were incubated with fresh plasma as source of LCAT. The esterification of cholesterol and cholesterol oxides was followed by measuring the consumption of the respective free sterol and oxysterols. Measurements of cholesterol and cholesterol oxides were done by gas chromatography with flame ionization detection. Results: All the studied cholesterol oxides were esterified by LCAT after incorporation into HDL particle, competing with cholesterol by LCAT-mediated esterification. Cholesterol esterification by LCAT was inversely related to the cholesterol oxide concentration in HDL particles. Kinetic studies with cholestan- 38, 5cr, 6B-trio1 and 5-cholesten- 3/I-25diol (25-hidroxycholesterol) showed a non-competitive inhibition with a Kiapparent of 103 and 15.02 ng/pL, respectively. This study shows markedly differences for inhibition parameters and apparent Ki among the different cholesterol oxides. Conclusions: Data suggest that cholesterol esterification by LCAT is inhibited in the cholesterol oxide-enriched HDL particles which could disturb the reverse cholesterol transport. In contrast, the esterification of cholesterol oxides by LCAT may have an anti-atherogenic effect considering that this step may increase the catabolism of these atherogenic compounds by enhancing their liver uptake either by direct HDL removal or by the previous transfer of the cholesteryl oxide esters to apo, B-containing lipoproteins followed by their hepatic catabolism. Supported by FAPESP

Conclusions: The hyperalphalipoproteinemia was explained, part, by the lower plasma levels of HL in this population.

I WeP32 W22

1 Lipases and transfer proteins in patients with hyperalphaiipoproteinemia

S. Alarcon’,V. Castanho’, D. Kaplan’, H. Oliveira*, V Nunes3, P. Cazita3, E. Quintio3, E. de Faria’ I Dept. of Clinical Pathology/NMCE/Il;NICAMP; 2Dept. of Physiology/UNICAM; 3Lipid Lab., FM-LISP Sr70 Paulo, Brazil Objective: To determine in Brazilian hyperalphalipoproteinemic (HALP) patients the activities of factors that modulate the metabolism of HDL: lipoprotein lipase (LPL), hepatic lipase (HL), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP). Methods: Ninety-five volunteers were defined by their HDL-chol as controls (CTL-n = 35, below 68 mg!dL) and HALP (n = 60, equal and above 68 mg/dL), the 90th percentile for a local population. HL, (post-heparin plasma), CETP and PLTP were measured by exogenous radiometric methods using respectively triolein emulsion, “C-CE-HDL and phospholipid lipossomes as substrates. Results: HL was significantly lower (p = 0.04) in HALP, but no differences were found for LPL, CETP and PLTP (Mann-Whitney). The scatterplots are shown below:

HALP

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3

CT1

LCAT-dependent cholesterol esterhkation rate is slower in in vi&o giycated HDL but is not altered in plasma HDL drawn from NIDDM patients

M. Passarelli, E.R. Nakandakare, S. Catanozi, J.C. Rocha, S.A. Lottenberg, E.C.R. Ouinfio. Lipids Lab. (LIM IO), Univ. S. Paul0 Med. School, S. Paulo, SP Brazil Objective: Alterations in the reverse cholesterol transport has been related to glycation of plasma lipoproteins. Present study investigates the influence of HDL glycation on the in vitro activity of the enzyme lecithin cholesterol acyl transferase (LCAT). Methods: HDL obtained from normal controls (N) and poorly controlled NIDDM (D) patients was separated by ultracentrifugation, and plasma d > 1.21 g/mL from N was utilized as the source of LCAT. Reconstituted HDL (ret HDL) from N were prepared by sonication of delipidated HDL previously submitted to selective glycation of the lipid and protein components separately, or both together, by exposure to a concentrated glucose solution. RecHDL as well as intact HDL from N and D were then labeled with “C-unesterified cholesterol (14C-C) and incubated with LCAT at 37°C along time. 14C-C ester and 14C-C were isolated after TLC and the percent of esterification was calculated. Results: The in vitro glycation of the intact HDL N particle, as well as of its protein, but not of its lipid component, impaired the activity of plasma LCAT. However, plasma LCAT esterified equally well 14C-C HDL that had been drawn from N (n = 11) and from D patients (n = 12). In spite of the elevated level of HBA’, in D, there was no difference in the level of HDL glycation between N and D subjects. Conclusion: In vitro HDL particle glycation rate impairs the LCAT-dependent 14C-C esterification rate. However, this rate is not modified in HDL D as compared to HDL N plasma, possibly because the faster removal rate of HDL D brings on a low plasma glycated HDL level.

I WeP33 W22 1WeP31 :W22

at least in

The stimuiation of cholesterol efilux by cpt-CAMP and free apo A-I from various cell lines

A.E. Bortnick, G.R. Rothblat, G. Stoudt, K.L. Hoppe’, L. Royer’, 0. Francone’. MCP Hahnemann University, Dept. of Biochem., 2900 Queen Lane, Phila., PA 19129; ‘PJizer Inc., Dept. of Cardiovascular and Metabolic Diseases, Central Research Division, Groton, CT, 06340, USA Studies have shown that unassociated, lipid-free apo A-I stimulates the release of cholesterol and phospholipid from cells. ATP-Binding Cassette 1 (ABCl) is implicated in this release, providing evidence that it is critical in the formation of HDL. Specific binding of apo A-I is upregulated by CAMP or enrichment with cholesterol. In this study, we determined the kinetics of cholesterol efflux from 5774 mouse macrophages with and without I exposure to cpt-CAMP. Upregulation is correlated to increased cholesterol efflux in a dose- and time-dependent manner with efflux first detectable 2-4 hours after treatment with cpt-CAMP. Efflux is upregulated by cholesterol enrichment of the same cell line. Stimulated efflux exhibits specificity for apo A-I, HDL, apo E and apo C as acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin, demonstrating that a protein-specific interaction is required. We have extended this study to 13 other cell lines under a standardized protocol, including fibroblasts (normal, transformed, and sitosterolemic), human and murine macrophages, hepatocytes, kidney cells, ovarian cells, and human enterocytes. Only J774 land elicited mouse macrophages show a significant increase in efflux with treatment. Apo A-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment. Apo A-I-stimulated efflux varied greatly among cell types, both in the percent and calculated mass of sterol released. Other data are consistent with upregulation of ABC1 by cpt-CAMP and cholesterol enrichment.

HALP

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WeP34.W22

Cholesteryl ester transfer protein activity and hyperalphalipoproteinaemia in Chinese

K.C.B. Tan, S.W.M. Shiu, E.D. Janus’. Department of Medicine, University of Hong Kong, Hong kong,China;Melboume Lipid Clinic, Melbourne, Australia 4

CTL

HALP

Objective: Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and plays a significant role in HDL metaboKHth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 20130