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Characteristics of ten LDL subfractions isolated by ion-exchange chromatography
220
Wednesday 12 October 1994: Poster Abstracts Lipoproteins
A rapid and simple method for the quantitative determination of serum apo E-rich HDL is reported. The sample is divided into two, one part being mixed with an equal volume of 13% polyethylene glycol6000, and the other with a commercial reagent consisting of dextran sulfate, sodium phosphotungstate and Mg2+. The solutions arc centrifuged. The supemate from the first procedure contains both apo E-rich and apo E-poor HDL, and that from the second procedure contains only apo E-poor HDL. The apo Erich HDL cholesterol concentration is obtained by subtraction. The serum apo E-rich HDL cholesterol concentration in 38 normal subjects was 6.7 f 2.3 mg/dl (mean f SD). It was as high as 50-100 mgldl in cases of intrahepatic cholestasis (2 cases of primary biliary cirrhosis and 1 of chronic hepatitis). In two cases of extrahepatic cholestasis (biliary tract malignancy), it was 3050 mg/dl and decreased promptly after bile drainage. Thus, a close relationship between apo E-rich HDL metabolism and bile excretion was shown by this method.
non-Hispanic whites. There was no significant difference in age, body mass index, or HDL cholesterol by apo E phenotype. Subjects with apo QR (n = 34) had a less athero enic profile (TG 140 mg/dl, LDL-C 223 mg/dl, LDL size 260.9 1 ) than subjects with apo E3 (n = 310) (TG 148 mg/dl, LDL-C 145 mg/dl, LDL size 256.8 R ) or apo Es/4 (n = 59) (TG 185 mg/dl, LDL 147 mg/dl, LDL size 252.8 A) (P values 0.010, 0.012 and
Role of vascular endothelial cells plated on culture dishes and polyurethane patch in in&unmation: an in vitro studv Maiwald G, Meyer G, Walli A, Schildberg FW, Seidel D, Dept. of
Characteristics of ten LDL subfractions isolated by 11541 Chappeyig:;yhange chromatography2 Myara Ilv2, Benott MO , Mazitre C3, Maxi&e X3, Moatti N1*2, I Lab. de Biochimie, Fact& des Sciences
Surgery, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, dermany
Pharmaceutiques et Biologiques, F-92296 Chatenay-Malabry Cedex; *Lab. de Biochimie, HGpital Broussais, F-75674 Paris Cedex 14; 3Lab. de Biochimie, CHU Saint Antoine, 27 rue de Chaligny, F-75012 Paris, France
1
We cultured human endothelial cells (EC) from saphenous vein and pulmonary, mesenteric, and iliac arteries. Endothelial cells in second passage were seeded on transparent polyurethane patch (graft material). Cultured cells grown on the patch showed no differences in their morphology and the presence of factor VIII. We investigated the adhesion of the human myelomonocytic cell line U937 to these cells in the presence of LDL (= 100 pg cholesterol), oxLDL and bacterial lipopolysaccharide (LPS, 2pg/ml 18 h) compared to normal cultured cells. A significant increase of U937 adhesion was noted by the stimulation of EC with LPS, in contrast caused only 30% of adhesion compared to normal LDL and oxLDL. Slight differences were noted between the same cell types cultured on patch and without the patch. Immunohistochemical staining with monoclonal antibodies showed the expression of intercellular adhesion molecule (ICAM1) and vascular cell adhesion molecule (VCAM-1) by EC. Inflamed tonsil was used as a positive control in all experiments, and as a negative control, all monolayers were stained with 12.5pg/ml of MOPC21, which lacks relevant antigenic specificity. After stimulation with LPS, expression of ICAM- and VCAM-1 was significantly increased in EC grown in culture dishes or on patch. No differences were noted between different cell types. However, endothelin release decreased in cells cultured on patch. From our studies it appears that polyurethane is a suitable graft material for endotheliahzation. EC grown on this material maintain their morphology, the presence of factor VIII and their normal metabolic activities. Studies are in progress to examine the suitability of these grafts in vivo. LDL size and apo E isoform in the San Antonio Heart Study J-IaffnerSM, Valdez R, Stem M, Howard B, Dept. of Med., Univ.
1159)
of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 7X284-7873, USA
Apo E polymorphism has been associated with alterations in lipoproteins. Recently, small dense LDL particles have been associated with increased coronary heart disease risk (CHD). We examined the correlation of apo_E polymorphism with LDL size (in 8, by method of Krauss and Burke, JLR 1982) in 403 subjects from the San Antonio Heart Study, a population-based study of diabetes and cardiovascular disease in Mexican Americans and
Native plasma LDL were fractionated into ten subfractions with increasingly negative charges (LDL-1, the least electronegative, to LDL-10) using an anion-exchange column attached to a fast protein-liquid chromatography system. No significant difference in thiobarbituric reactive substances, vitamin E or free aminogroup content was found between subfractions. We observed a gradual decrease in triglycerides and a concomitant increase in cholesteryl esters from LDL-1 to LDL-6, and the reverse tendency from LDL-7 to LDL-10 (P < 0.05). An increase in free cholesterol and phospholipids was also found in LDL-8 to LDL-10 (P < 0.05). The LDL subfractions had a heterogeneous density distribution related to the lipid changes, and a slight increase in density was found in the most electronegative subfractions. Competitive binding curves obtained with labeled LDL-1 on HepG2 cells and fibroblasts showed that unlabeled LDL4 reduced the binding of radioactive LDL-1 to the same extent as unlabeled LDL-1. By contrast, unlabeled LDL-8 competed less actively with ‘251-labeled LDL-1 for receptor binding. Similar data were obtained with unlabeled LDL subfractions 1,4, 8 competing with labeled LDL-4. Higher concentrations of LDL-8 subfraction (about +50% and +30% on HepG2 cells and fibroblasts, respectively) than LDLl and LDL4 subfractions were required to induce 50% displacement of labeled LDL-1 and LDL-4, suggesting that this electronegative subfraction had a lower receptor binding affinity. In addition, subfractions LDL-6 to LDL-8 were most sensitive to copper-induced oxidation (41% decrease in lag time vs LDL-1 and 18% vs LDL-2 to 5). In conclusion, although most electronegative LDL subfractions are not oxidized, their lower binding to cells and highest susceptibility to oxidation suggest that the most electronegative subfractions may be most atherogenic, a hypothesis that is under investigation in populations with cardiovascular risk factors. Which is the most important lipid risk factor in peripheral atherosclerosis? Mijlgaard J, Olsson AG, Dept. of Int. Med., Univ. Hosp., S-581
1155(
85, Linkiiping, Sweden
Objective: To establish the relative importance of LDL-C, Lp(a), TG, HDL-C and apo E isoforms in peripheral atherosclerosis. 100 men with intermittent claudication (IC) and 100 healthy
Atherosclerosis X, Montreal, October 1994
Wednesday 12 October 1994: Poster Abstracts Lipoproteins
A rapid and simple method for the quantitative determination of serum apo E-rich HDL is reported. The sample is divided into two, one part being mixed with an equal volume of 13% polyethylene glycol6000, and the other with a commercial reagent consisting of dextran sulfate, sodium phosphotungstate and Mg2+. The solutions arc centrifuged. The supemate from the first procedure contains both apo E-rich and apo E-poor HDL, and that from the second procedure contains only apo E-poor HDL. The apo Erich HDL cholesterol concentration is obtained by subtraction. The serum apo E-rich HDL cholesterol concentration in 38 normal subjects was 6.7 f 2.3 mg/dl (mean f SD). It was as high as 50-100 mgldl in cases of intrahepatic cholestasis (2 cases of primary biliary cirrhosis and 1 of chronic hepatitis). In two cases of extrahepatic cholestasis (biliary tract malignancy), it was 3050 mg/dl and decreased promptly after bile drainage. Thus, a close relationship between apo E-rich HDL metabolism and bile excretion was shown by this method.
non-Hispanic whites. There was no significant difference in age, body mass index, or HDL cholesterol by apo E phenotype. Subjects with apo QR (n = 34) had a less athero enic profile (TG 140 mg/dl, LDL-C 223 mg/dl, LDL size 260.9 1 ) than subjects with apo E3 (n = 310) (TG 148 mg/dl, LDL-C 145 mg/dl, LDL size 256.8 R ) or apo Es/4 (n = 59) (TG 185 mg/dl, LDL 147 mg/dl, LDL size 252.8 A) (P values 0.010, 0.012 and
Role of vascular endothelial cells plated on culture dishes and polyurethane patch in in&unmation: an in vitro studv Maiwald G, Meyer G, Walli A, Schildberg FW, Seidel D, Dept. of
Characteristics of ten LDL subfractions isolated by 11541 Chappeyig:;yhange chromatography2 Myara Ilv2, Benott MO , Mazitre C3, Maxi&e X3, Moatti N1*2, I Lab. de Biochimie, Fact& des Sciences
Surgery, Klinikum Grosshadern, Marchioninistr. 15, 81377 Munich, dermany
Pharmaceutiques et Biologiques, F-92296 Chatenay-Malabry Cedex; *Lab. de Biochimie, HGpital Broussais, F-75674 Paris Cedex 14; 3Lab. de Biochimie, CHU Saint Antoine, 27 rue de Chaligny, F-75012 Paris, France
1
We cultured human endothelial cells (EC) from saphenous vein and pulmonary, mesenteric, and iliac arteries. Endothelial cells in second passage were seeded on transparent polyurethane patch (graft material). Cultured cells grown on the patch showed no differences in their morphology and the presence of factor VIII. We investigated the adhesion of the human myelomonocytic cell line U937 to these cells in the presence of LDL (= 100 pg cholesterol), oxLDL and bacterial lipopolysaccharide (LPS, 2pg/ml 18 h) compared to normal cultured cells. A significant increase of U937 adhesion was noted by the stimulation of EC with LPS, in contrast caused only 30% of adhesion compared to normal LDL and oxLDL. Slight differences were noted between the same cell types cultured on patch and without the patch. Immunohistochemical staining with monoclonal antibodies showed the expression of intercellular adhesion molecule (ICAM1) and vascular cell adhesion molecule (VCAM-1) by EC. Inflamed tonsil was used as a positive control in all experiments, and as a negative control, all monolayers were stained with 12.5pg/ml of MOPC21, which lacks relevant antigenic specificity. After stimulation with LPS, expression of ICAM- and VCAM-1 was significantly increased in EC grown in culture dishes or on patch. No differences were noted between different cell types. However, endothelin release decreased in cells cultured on patch. From our studies it appears that polyurethane is a suitable graft material for endotheliahzation. EC grown on this material maintain their morphology, the presence of factor VIII and their normal metabolic activities. Studies are in progress to examine the suitability of these grafts in vivo. LDL size and apo E isoform in the San Antonio Heart Study J-IaffnerSM, Valdez R, Stem M, Howard B, Dept. of Med., Univ.
1159)
of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 7X284-7873, USA
Apo E polymorphism has been associated with alterations in lipoproteins. Recently, small dense LDL particles have been associated with increased coronary heart disease risk (CHD). We examined the correlation of apo_E polymorphism with LDL size (in 8, by method of Krauss and Burke, JLR 1982) in 403 subjects from the San Antonio Heart Study, a population-based study of diabetes and cardiovascular disease in Mexican Americans and
Native plasma LDL were fractionated into ten subfractions with increasingly negative charges (LDL-1, the least electronegative, to LDL-10) using an anion-exchange column attached to a fast protein-liquid chromatography system. No significant difference in thiobarbituric reactive substances, vitamin E or free aminogroup content was found between subfractions. We observed a gradual decrease in triglycerides and a concomitant increase in cholesteryl esters from LDL-1 to LDL-6, and the reverse tendency from LDL-7 to LDL-10 (P < 0.05). An increase in free cholesterol and phospholipids was also found in LDL-8 to LDL-10 (P < 0.05). The LDL subfractions had a heterogeneous density distribution related to the lipid changes, and a slight increase in density was found in the most electronegative subfractions. Competitive binding curves obtained with labeled LDL-1 on HepG2 cells and fibroblasts showed that unlabeled LDL4 reduced the binding of radioactive LDL-1 to the same extent as unlabeled LDL-1. By contrast, unlabeled LDL-8 competed less actively with ‘251-labeled LDL-1 for receptor binding. Similar data were obtained with unlabeled LDL subfractions 1,4, 8 competing with labeled LDL-4. Higher concentrations of LDL-8 subfraction (about +50% and +30% on HepG2 cells and fibroblasts, respectively) than LDLl and LDL4 subfractions were required to induce 50% displacement of labeled LDL-1 and LDL-4, suggesting that this electronegative subfraction had a lower receptor binding affinity. In addition, subfractions LDL-6 to LDL-8 were most sensitive to copper-induced oxidation (41% decrease in lag time vs LDL-1 and 18% vs LDL-2 to 5). In conclusion, although most electronegative LDL subfractions are not oxidized, their lower binding to cells and highest susceptibility to oxidation suggest that the most electronegative subfractions may be most atherogenic, a hypothesis that is under investigation in populations with cardiovascular risk factors. Which is the most important lipid risk factor in peripheral atherosclerosis? Mijlgaard J, Olsson AG, Dept. of Int. Med., Univ. Hosp., S-581
1155(
85, Linkiiping, Sweden
Objective: To establish the relative importance of LDL-C, Lp(a), TG, HDL-C and apo E isoforms in peripheral atherosclerosis. 100 men with intermittent claudication (IC) and 100 healthy
Atherosclerosis X, Montreal, October 1994