118 Tuesday 7 October 1997: Posters Lipid-lowering drugs antibody for detection of lipoproteins after electrotransfer, we have routinely detected up to five species of y-LpE in human plasma, ranging from 9.5 to 16.5 nm in mean particle diameter. The majority of y-migrating apoE, representing less than 5% of total plasma apoE, was associated with y2-LpE having a diameter of 13.0 mu. Neither the amount of y-LpE apoE nor the number of y-LpE species was different in serum v plasma or was affected by the presence of agents able to inhibit protein dimerization during plasma preparation. Incubation of plasma at 37°C (90 min) caused a significant decrease in plasma y-LpE (>50%), which was not dependent on LCAT or CETP activity. Incubation of plasma with cultured fibroblasts (37°C 1 min) caused a significant increase (48 f 14%. mean f SD) in plasma y-LpE @ < 0.05). Storage at -70°C of hypertriglyceridemic but not normolipidemic plasma resulted in an increase in y-LpE. Freezing of postprandial plasma samples, containing increased amounts of triglyceride-rich lipoproteins rich in apoE, also resulted in an increase in y-LpE. Incubation of VLDL (d < 1.006 g/ml) with lipase in vitro caused the production of y-LpE-like particles. These results demonstrate that: 1) different-sized y-LpE particles exist in human plasma; 2) decrease in plasma y-LpE concentration due to incubation at 37°C is not due to LCAT or CETP activity and is reversed by the presence of cells, and 3) y-LpE can be produced in vitro from plasma VLDL. El 2.P.8 Mutations of the branchpoint concensus sequence of intron 4 of lecithin: Cholesterol acyltransferase (LCAT) gene M., PH. Pritchard. Vniversit) of British Columbia, Vancouvtx Canada We have previously studied a single nucleotide substitution (T-+C) in a branchpoint region of intron 4 of LCAT in a family with fish-eye disease (FED) which causes a null allele as the result of complete intron retention. In order to further study the functional significance of the intron branchpoint sequence, two new mutations, intron 4 MUT-1 (T-+G) and MUT-2 (T-+A), were introduced into the same site in which the natural mutation occurs, and then cloned into the mammalian vector pNUT. After the pNUT-LCAT mini- genes were stably transfected into BHK cells, the activity and concentration of LCAT in the culture medium were determined. The specific activity for LCAT cDNA and the intron 4 wild type (2.34 f 0.68 vs. 2.23 f 1.21 nmol/h/wg) was nearly the same. However, no LCAT activity and protein was detected for the imron 4 MUT-1 and MUT-2, and the natural intron 4 mutation. RT-PCR revealed that LCAT expression was evident in LCAT cDNA, intron 4 wild type, and the intron 4 mutants. However, the sizes of these RT-PCR products for LCAT cDNA, the intron 4 wild type and the mutants are different. The difference between these fragments was 83 bp in length, indicating that the intronic sequence was retained. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the desired mutations. In conclusion, the loss of the T, two bases upstream of the internal adenosine residue in the putative branchpoint consensus sequence of the intron 4 of LCAT gene, disrupts the process of pre-mRNA splicing and results in the intron retention. Ll 2 P 7 Age-related decline in plasma CETP and CETP mRNA expression in human adipose tissue T. Radeau, M. Robb, P. Lau, J. Borthwick, R. McPherson. Vniversity of Ottawa Heart Institute, Ottawa, Canada Adipose tissue is an important site of CETP synthesis. However, it is not known to what extend adipose tissue CETP contributes to plasma pool of CETP in man Aging is often associated with changes in adipose tissue composition and function. We have determined adipose tissue CETP mRNA concentration by RNase protection assay and plasma levels of CETP by radio-immunoassay as well as plasma lipoproteins in 12 middle aged and elderly subjects with stable coronary heart disease at the time of coronary artery bypass surgery. Plasma concentrations of CETP were highly correlated with pericardial adipose tissue CETP mRNA abundance (R = 0.89, P < 0.002). CETP mRNA concentrations in adipose tissue were inversely related to age (R = -0.72, P < 0.03) as were plasma concentrations of CETP (R = -0.69, P < 0.012). These relationships were independent of plasma lipids, lipoproteins and body mass index. However, adipocyte size, estimated by adipose tissue triglyceride/mg soluble protein were related to CETP mRNA levels (R = -0.76, P < 0.02). There was also a trend for increased adipocyte size with aging. The age-related decline in plasma concentrations of CETP can be explained in part by a decrease in adipose tissue CETP gene expression. I 2 P.8 Expression of CETP mRNA by stromal vascular cells of human ndipose tissue T. Radeau, M. Robb, M. McDonnel, R. McPherson. Vniversity of Ottawa Heart Institute, Ottawa, Canada CETP is highly expressed in human adipose tissue and its expression may be modulated by fat cell size. Stromal vascular fraction of adipose tissue is com- posed of various cell types including immature adipocytes, pre-adipocytes, macrophages and endothelial cells. In this study, the relative expression of CETP mRNA was first investigated in mature fat cells (MFC) and stromal- vascular cells (SVC). By Northern blot analysis, CETP: 18 S RNA ratio was significantly higher in SVC as compared to MFC (2.18 f 0.21 vs 1.13 & 0.18, 1’ < 0.03, N = 4) whereas lipoprotein lipase/ll S RNA ratio was significantly lower (3.6 f 0.57 vs 14.1 & 2.8, P < 0.03). Pre-adipocytes were further purified from SVC by adherence on plastic and mRNA expression determined by RT-PCR. When normalized to fi actin, CETP mRNA expres- sion in pre-adipocytes was only 20% of that found in SVC. Moreover, the presence of a significant number of monocytes/macrophages was excluded since CD16 mRNA, a marker of these cells, was not found in MFC, SVC or pre-adipocytes. We also demonstrated that a human endothelial cell line does not (express CETP mRNA. These data suggest that a major part of CETP mRNA ir human adipose tissue is produced by SVC, possibly by immature or very small fat cells. cl 2 P 9 Reduced phwma HDL-cholesterol levels in visceral obesity: Contribution of the CETP gene TaqIB polymorphism M.C. Vohl, B. Lamarche, G. Leroux, D. Prud’homme, C. Bouchard, A. Nadeau, J.P. Desprt?s. Lipid Research CenteK Diabetes Research Unit and LABSAP, Lava1 University Ste-Fey, QuPbec. Canada The aim of the present study was to examine whether the TaqIB polymorphism of the CE.TP gene influence HDL-cholesterol levels in visceral obesity. A total of 135 sedentary men: 1) 47 BlBl homozygotes (HMZ) for the presence of the restriction site, 2) 54 BlB2 heterozygotes (HTZ) and 3) 34 B2B2 HMZ participated in this study. The three groups were similar for age, BMI, and vis- ceral adipose tissue (VAT) accumulation assessed by computed tomography. However B2B2 men had significantly higher HDL and HDLs-chol levels than BlBl HMZ (p < 0.05). In order to determine the source of variation of plasma HDL-chol levels, the three genotypic groups were divided into low vs high (< or > 130 cm2) VAT accumulation. The amount of VAT explained 7.6% and 9.4% of he variation in plasma HDL and HDLz-chol levels (p < 0.001). No significant effect of the genotype was observed on HDL-chol levels when VAT accumulation was taken into account. The CETP-TaqIB genotype explained 4.5% of the variation in plasma HDLZ-chol levels (p < 0.05). Essentially similar results were obtained when subjects were divided on the basis of the median value of fasting insulin levels, the CETP-TaqIB genotype explaining 9.3% (p < 0.01) of the variation in plasma HDLZ-chol levels. These results suggest that the CETP-TaqIB polymorphism influences the magnitude of the decrease in plasma HDL-chol levels observed in visceral obesity. LIPID-LOWERING DRUGS II 2.P.10 Effects of erythromycin on plasma fluvastatin levels: A pharmacokinetic study J. Aberg U. Eriksson, G. Fager. Clinical Pharmacology, Clinical Research and Development, Astra Hiissle AB, Miilndal, Sweden Introdu&ion: Fluvastatin (F) is a new hydroxy-methylglutaryl coenzyme A reductase inhibitor. At daily doses of 20 to 80 mg it provides marked reduc- tions of LDL cholesterol similar to other drugs of this class. F is predominantly eliminated by hepatic metabolism and it is therefore important to investigate the potential interaction with other drugs eliminated by hepatic metabolism. Erythromycin (E) is cleared by the liver and a suitable model for cytochrome P450 3A depending drugs. Aim: To study the pharmacokinetics of steady state plasma concentrations of F following concurrent administration of a single dose of E. Study Design: The study was an open, randomized, two-way, cross-over design. Twenty-four healthy male subjects (age 21-34 years) participated. Subjects were given F capsules 40 mg o.d. for 12 days. On day 6 and 12 the subjects were randomly given E tablets 500 mg as a single dose or no E. Blood for F plasma assay was frequently drawn on days 6 and 12. 11th International Symposium on Atheroscleroris, Paris, October 1997