4.P.365 Increased capacity of cellular cholesterol efflux of serum after moderate intake of red wine in young men

Thursday 9 October 1997: Poste, s HDL/reverse cholesterol transport gene in mouse ES cells . Homozygous LCAT deficient (LCAT- ± ) mice were viable and fertile . Plasma LCAT activity in age-matched control siblings (n = 9, LCAT act = 44 f 3 nmol/h/ml) was decreased to 30 ± 2 nmol/h/ml (p < 0 .01) in heterozygous LCAT deficient mice (n = 13) and to 0 .3 ± 0.1 mnol/h/ml (p < 0 .01) in LCAT- ± mice (n = 11). Compared to control mice with TC = 134 ± 22 mg/dl, TG = 109 ± 23 mg/dl, PL = 244 ± 39 mg/dl, CE = 106 ± 18 mg/dl, HDL-C = 92 ± 17 mg/dl and apoA-I = 99 ± 19 mg/dI, LCAT- ± mice had a decrease (% of control, p < 0 .01) in TC (23%), CE (11%), PL (41%), HDL-C (7%), apoA-I (14%) and an increase in TG (212%) . Analysis of plasma lipoproteins in LCAT- ± mice by FPLC demonstrated severe reduction in HDL-C with the presence of smaller sized particles, as well as TG-rich IDL/LDL. Two-dimensional gel electrophoresis of plasma lipoproteins in LCAT- ± mice demonstrated the presence of heterogeneous prep-HDL particles . After 3 weeks on a high-fat high-cholesterol (HFHC) diet, LCAT- ± mice had significantly lower plasma concentrations of TC, reflecting reduced levels of both proatherogenic apoB-containing lipoproteins as well as HDL, compared with controls . No evidence of corneal opacities or renal insufficiency was detected in 8-month-old LCAT- ± mice. In summary: 1) We have generated LCAT deficient mice by gene targeting in ES cells . 2) LCAT-deficient mice have marked reductions in TC, CE, PL, HDL-C and apoA-I as well as increased TG and heterogeneous pre #-HDL particles, a lipoprotein phenotype similar to that of human patients . 3) LCAT modulates the mouse lipid response to a HFHC diet in a dose-dependent manner with increasing LCAT activity. 4) The availability of a homozygous animal model for human LCAT deficiency will permit analysis of the role of LCAT in atherosclerosis as well as the feasibility of performing gene transfer in human LCAT deficiency states.

4 .P.363

Two novel mutations of cholesteryl ester transfer protein (CETP) gene in Japanese hyperalphalipoproteinemic patients

N . Sakai, T. Arai, T. Maruyama, S . Yamashita, S . Santamarina-Fojol, H .B . Brewer, Jr.', Y. Matsuzawa. 2nd Dept Int Med, Osaka Univ Med School, 2-2 Yamadaoka, Suita, Osaka 565, Japan; 'MDB, NHLBI, NIH, MD, USA Cholesteryl ester transfer protein (CETP) plays an important role in HDL metabolism and reverse cholesterol transport . Patients with CETP deficiency present with marked hyperalphalipoproteinemia (HALP) . CETP deficiency is the most common cause of HALP in Japan . Two common mutations (intron 14 splice defect and D442G) have been identified in these patients and account for approximately 50% of HALP caused by decreased CETP activity . However, the genetic basis of this disorder has not been fully characterized . We investigated the underlying molecular defect in the CETP gene of two patients presenting with lipoprotein abnormality of this genetic disorder. The patients were a 49-year-old Japanese male and 62-year-old Japanese female . Their plasma HDL-C levels were 236 mg/dl and 153 mg/dl, respectively . Sequence analysis of the first patient revealed that the splice donor consensus GT was substituted by GG in intron 10 (intron 10 splice defect) and by AT in intron 14 (intron 14 splice defect) . Sequencing of cDNA amplified by RT-PCR from the patient's monocyte-derived macrophage RNA demonstrated abnormal splicing with deletion of exon 10 as well as alternative splicing at a native AG site located 31 nucleotides 5' of the normal splice acceptor in intron 13 . This inserted 31 by sequence contained an in frame stop codon . The second patient was also a compound heterozygote for the intron 14 splice defect and a G-to-T substitution at codon 181 of exon 6 (G181X), resulting in the formation of a premature stop codon . PCR-RFLP was used to screen 294 HALP patients (HDL-C > 100 mg/dl) for these two new mutations . One (0.34%) homozygote and four (1 .36%) heterozygotes for the G181X mutation were found, while intron 10 splice defect was not found at all . In summary 1) We found two novel CETP mutations (intron 10 splice defect and G181X) . 2) Intron 10 splice defect resulted in the disruption of downstream splicing at intron 13, a novel mechanism leading to CETP deficiency . 3) The allelic frequency of a G181X mutation was 0.0102 in Japanese HALP patients but intron 10 splice defect was not found in other HALP .

4 .P.364

Effects of hypochlorte-modification on physico-chemical and biological properties of high density lipoproteins

W. Sattler, S . Raitmayer, G . Kostner, Ernst Malle, U . Panzenboeck . University Graz, Department of Medical Biochemistry, Graz, Austria During the present study the effects of hypochlorite - one of the oxidants generated by the myeloperoxidase/hydrogen peroxidetchloride system - on physico-chemical and metabolic properties of HDL (subclass 3, HDL3) were investigated. During in vitro NaOC1 (OCl- ) modification of HDL3 we have

373

observed pronounced alterations in the apolipoprotein- and the lipid domain. Exposure of HDL3 to increasing NaOCI concentrations resulted in the oxidation of amino- and fatty acids . Up to a molar OCl- :HDL3 ratio of 30:1 apo A-I appeared to represent the preferential target for OCl - attack (consuming 35-76% of the oxidant), thereby providing good (though no absolute) protection for HDL 3 fatty acids (consuming between 17 and 30% of the oxidant) against OCI- mediated modification . At oxidant: HDL3 molar ratios >60:1 we have observed pronounced modification of unsaturated fatty acids with the concomitant formation of fatty acid chlorohydrins . Presumably due to the modifications in apo A-I HDL 3 -metabolism by mouse peritoneal macrophages was altered significantly : Native HDL3 lost its ability to compete for uptake of OCI - modified HDL3 . In addition HDL3-holoparticle turnover (binding, internalization and degradation) and the selective internalization of HDL3-associated cholesterylesters was enhanced up to 4 fold and resulted ultimately in the formation of lipid laden foam cells as verified by fluorescence microscopy . Finally, the ability of HDL3 to promote cellular cholesterol efflux from macrophages was significantly diminished (2 fold increase in r/2 values) after OCl - modification . Decreased cholesterol efflux promoting abilities of modified HDL3 preparations were clearly dependent on the rate of OCl modification . Collectively, these results demonstrate that the modification of HDL3 with OCl - transformed an antiatherogenic lipoprotein particle into a modified lipoprotein with characteristics similar to lipoproteins commonly thought to initiate foam cell formation in vivo.

4 .P.365

Increased capacity of cellular cholesterol efflux of serum after moderate intake of red wine in young men

C . Senault, I). Betoulle, G . Lucl , F. Fumeron. Laboratoire de Nutrition Humaine, Faculte Xavier Bichat, Paris ; t INSERM U325, Lille, France Moderate alcohol consumption has been shown to be protective against cardiovascular disease. This effect is thought to be partly mediated through an increase of The levels of high density lipoproteins (HDL) . Red wine has been claimed to be more protective than other alcoholic beverages . The aim of our work was to assess the effect of red wine on the reverse cholesterol transport parameters including HDL related variables and cellular cholesterol efflux . Thirty sixx young men drunk during 14 days either red wine or alcoholic solution (31 g alcohol/day) . Each beverage was tested in a random order after a 2 week period of total abstinence from alcohol. Red wine consumption increased significantly serum HDL-C (p = 0 .0001), ApoAI (p = 0 .001), HDL3-C (p = 0 .001), LpAI and LpAI All particles (p = 0 .046 and 0.044) . Plasma CETP plasmatic concentrations remained unchanged . Alcoholic solution effects were less significant than those of red wine : only ApoAl (p = 0 .012) and LpAI All (p = 0.008) increased. Serum promoted cholesterol efflux from Fu5AH cells was measured in 12 subjects . It was increased by 5% (p = 0 .001) only after red wine. In the abstinent state, cholesterol efflux was positively correlated with ApoAl (p = 0 .012), LpAI All levels (p = 0 .008) and with HDL phospholipid and cholesterol composition . Variation in efflux was positively correlated with HDL-C (p = 0 .019), ApoAl (p = 0 .032), HDL3 (p = 0 .036) and LpAI All (p = 0 .006) variations . In this study, specific beneficial effect of red wine consumption was evidenced by increase of HDL-C and related parameters. The increase of the serum capacity to induce in vitro cellular efflux confirms that protective effect of wine could act through the first step of reverse cholesterol transport and that the HDL3 and LpAI All particles could be the main determinants of the cholesterol efflux potential .

4 .P.366

Characterization of two distinct hyperalphalipoproteinemic (HALP) profiles without cholesteryl ester transfer protein (CETP) deficiency

D. Sicha, Y. Saidi 1 , I . Beuclert, P. Gira1 2 , C . Cherfils a , M . Egloff1 , G. Turpin2 . t Laboratoire des Lipides; 2Service d'Endocrinologie, Hopital de la Pitie, 75651, Paris To evaluate the cardiovascular effect of elevated HDL-Cholesterol levels in presence of hypercholesterolemia, 29 patients with both elevated LDL-Cholesterol (180 == 35 mg/dL) and HDL-Cholesterol (90 ± 11 mg/dL) levels were investigated . The patients (mean aged: 55 ± 9 years) were asymptomatic for coronary heart disease despite high LDL levels . Ultrasonography of carotid arteries have more precisely characterized their cardiovascular state : 18 patients were free from lesions, 7 had intimal thickening and 4 displayed arterial plaque . The HDL subclasses were separated according to their density, size and apolipoprotein content . Two HALP profiles were defined according to the HDL2/HDL .3 and LpA-I/LpA-I:A-II ratios .

11th International Symposium on Atherosclerosis, Paris, October 1997