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Hydrophobic interactions in plasma proteins and pathogenesis of atherosclerosis
Poster session abstracts/Atherosclerosis 115 (Suppl.) (1995) $45-$129 PI8 Lipoproteins
SI23
461
463
INDICES OF LDL PARTICLE SIZE CLOSELY RELATE TO TRIGLYCERIDE LEVEL IN CORONARY ARTERY DISEASE ANGIOGRAPHICALLY DOCUMENTED R. Fermtndez and J. Pedrefio Dept Biochemistry. Hospital Son Dureta. Palma de Majorca. Spain
EVALUATION OF SIGMA AND IMMUNO METHODS FOR DIRECT MEASUREMENT OF LOW DENSITY LIPOPROTEIN CHOLESTEROL N. Capps, N. Parry Dept of Clinical Biochemistry, Princess Royal Hospital NHS Trust, Telford, UK
In addition to the evidence for increased coronary disease risk associated with the small, dense LDL particles, cross-sectional and prospective studies have also demonstrated that LDL particle size is negatively associated with plasma triglyceride level and positively with HDL 2 cholesterol level. Recently, (Dyce et al. Circulation 1993.88:1-466) it has been reported that LDL cholesterol:plasma apoB ratio is an index to assess the LDL particle size. The aim of this study was to evaluate the association between this ratio and both triglyceride and H D ~ cholesterol levels in 158 men with severe coronary artery disease (CAD) angiographlcally documented and to relate with the involvement of one or more vessels. The ratio was lower in CAD patients with a triglyceride level greater than 160 mg/dl compared to CAD patients with a triglyceride level below 160 mg/dl (0.98_+0 19 and 1.18±0.22. respectively), although this difference did not reach statistical significance. In univariate analysis the ratio in hypertriglyceridemic CAD patients associated with plasma triglyceride concentration (r=-0.47) and HDI< cholesterol (r=0.39), however not significant association was found in normotriglyceridemic patients. Multiple linear regreslon analysis showed that triglyceride level significantly predicted the ratio, but HDL2 cholesterol level was not a significant prethctor. When CAD-patients were divided according to triglyceride level (160-200 mg/dl, 200-300 mg/dl, and >300 mg/dl), a nonsignificant trend to decrease in ratio and to increase in the prevalence of values below 1.0 were found in more hypertriglyceridemic patients. Finally, the ratio showed significant lower values (p <0.05) in 2-and 3-vessel disease groups compared to those obtained in l-vessel group (1.16+0.24 in the single group. 1.05-t:0.22 in the 2-vessel group and 1.03+0.21 in the 3-vessel group). Moreover. there was a significant inverse association between the ratio and triglyceride level (p<0.0001) and a significant positive association with HDI< cholesterol level ~ < 0 . 0 0 1 ) in all groups. These results suggest that LDL cholesterol:plasma apoB ratio is strongly associated with triglyceride and HDL: cholesterol in CAD-patients. and seems to be a better discri minstor for the extent of coronary artery disease angiographically defined.
A new direct LDLC method, based on immunoseparation (IS) has recently been reported (Sigma Diagnostics) and we have evaluated this along with another technique based on selective LDLC precipitation (P) (Immuno Ltd), in particular examining the effects of high triglyceride and nonfasting samples when the Friedwald formula (Ff) becomes invalid. Correlations with calculated LDLC and apoB (lncstar) and stability studies were also performed. All cholesterols and triglycerides (TG) were performed on an Ektachem 250 and ApoB on a Cobas Fara. 22 pairs of fasting and nonfasting samples were obtained and TG in the form of intralipid (Clintec) dilutions was added to 25 fasting sera to increase concentrations by up to 30 mM (2700 mg/dl). 3 sera were analysed each day after storage at 2-8 and -20 degrees C for 10 days. Fasting LDLC was 3.92 (SD 1.20) by IS and 5.27 (1.16) by P, nonfasting 3.91 (1.[6) and 5.05 (1.21). No significant trends in LDLC were observed with either method with the addition of increasing TG concentrations and samples appeared stable at both temperatures for up to 10 days. Correlation coefficient (n=20) between LDLC by IS and P was 0.89, between IS and calculated 0.80, P and calculated 0.71, and between apoB and IS 0.77, ApoB and P 0.55 and Apo B and calculated 0.70. Both direct LDLC methods appear promising, particularly where Ff cannot be used with nonfasting samples and when TG is > 4 . 5 mM(170 mg/dl).
462
464
HYDROPHOBIC INTERACTIONS IN PLASMA PROTEINS AND PATHOGENESIS OF ATHEROSCLEROSIS B. Lipinski H.S. Research Laboratory, Cambridge MA 02139, U.S.A
A NEW METHOD FOR THE RAPID SEPARATION OF PLASMA LIPOPROTEINS J.M. Graham, J.A. Higgins, T. Gillot School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK; Dept of Molecular Biology, University of Sheffield, Sheffield SI0 2TN, UK; Northern General Hospital, Sheffield $5 7AU,UK.
Native plasma proteins are characterised by their relative hydrophilicity which prevents undesirable protein-protein and protein-cell interactions. Hydrophobic regions of proteins are normally buried in the interior of proteins and are stabilised by the intramolecular disulfide (S-S) bonds. These regions can be exposed in proteins by limited proteolysis and/or unfolding of the polypeptide chains resulting in a S-S exchange from intra- to intermolecular bonds. Limited reduction of human serum albumin (HSA) (e.g. with ascorbic acid) resulted in the formation of soluble hydrophobic multimers. A similar hydrophobic macromolecular protein complex (MPC) was identified in human plasma and was shown to be significantly increased in subjects with atherosclerosis. A characteristic feature of MPC is its ability to become incorporated into the fibrin clot during clotting of fibrinogen with thrombin. Fibrin clots obtained from the patient with atherosclerosis contained not only 50-80 % of fibrinogen-unrelated protein, but were also completely resistant to fibrinolysis induced with plasminogen activators. Incorporation of MPC into fibrin clots can be inhibited by certain amphiphilic drugs such as aurin tricarboxylic acid and suramin by virtue of the interaction of their hydrophobic moieties with those of fibrin monomers. Increased concentration of MPC in blood may thus contribute to pathogenesis of atherosclerosis making fibrin deposits refractory to fibrinolysis.
Variations in the relative amounts of plasma lipoprotein classes and/or molecular changes in their lipid components and apoproteins, are important factors in the development of atherosclerosis, A new equilibrium centrifugation method, which avoids high salt gradients, provides high resolution and concentration of lipoproteins for electrophoretic, compositional and structural analysis. The method uses a new non-ionic medium, Iodixanol, available as a 60% (w/v) solution called OptiPrep (Nycomed Pharma AS, Oslo). Plasma is adjusted to 12.5% lodixanol; overlaid with 0.1-0.2x its volume of saline and centrifuged at approx 350,000g,v for 2.5-3 h in a vertical or near-vertical rotor. Using 3.9 ml tubes, unloaded dense-end first; soluble proteins are contained in the first 1.0 ml, HDL in the 1.2 - 2.4 ml fraction; LDL in the 2.6 - 3.4 ml fraction and VLDL in the 3.6 - 3.9 ml fraction. There is little overlap between the three classes. Fractions can be analysed directly, by gel electrophoresis and for lipid content. The method may resolve different sub-classes of these lipoproteins; this is currently being investigated. Because of the concentration effect achieved by the gradient, Lp(a), which is recovered in a single 0.25 ml fraction, can be easily demonstrated on gels when levels in the whole plasma are below the limits of detection. The technique thus provides a means of fractionating and concentrating all plasma lipoproteins in a single step: it should prove to be a valuable diagnostic and analytical tool in studies of lipoprotein metabolism.
SI23
461
463
INDICES OF LDL PARTICLE SIZE CLOSELY RELATE TO TRIGLYCERIDE LEVEL IN CORONARY ARTERY DISEASE ANGIOGRAPHICALLY DOCUMENTED R. Fermtndez and J. Pedrefio Dept Biochemistry. Hospital Son Dureta. Palma de Majorca. Spain
EVALUATION OF SIGMA AND IMMUNO METHODS FOR DIRECT MEASUREMENT OF LOW DENSITY LIPOPROTEIN CHOLESTEROL N. Capps, N. Parry Dept of Clinical Biochemistry, Princess Royal Hospital NHS Trust, Telford, UK
In addition to the evidence for increased coronary disease risk associated with the small, dense LDL particles, cross-sectional and prospective studies have also demonstrated that LDL particle size is negatively associated with plasma triglyceride level and positively with HDL 2 cholesterol level. Recently, (Dyce et al. Circulation 1993.88:1-466) it has been reported that LDL cholesterol:plasma apoB ratio is an index to assess the LDL particle size. The aim of this study was to evaluate the association between this ratio and both triglyceride and H D ~ cholesterol levels in 158 men with severe coronary artery disease (CAD) angiographlcally documented and to relate with the involvement of one or more vessels. The ratio was lower in CAD patients with a triglyceride level greater than 160 mg/dl compared to CAD patients with a triglyceride level below 160 mg/dl (0.98_+0 19 and 1.18±0.22. respectively), although this difference did not reach statistical significance. In univariate analysis the ratio in hypertriglyceridemic CAD patients associated with plasma triglyceride concentration (r=-0.47) and HDI< cholesterol (r=0.39), however not significant association was found in normotriglyceridemic patients. Multiple linear regreslon analysis showed that triglyceride level significantly predicted the ratio, but HDL2 cholesterol level was not a significant prethctor. When CAD-patients were divided according to triglyceride level (160-200 mg/dl, 200-300 mg/dl, and >300 mg/dl), a nonsignificant trend to decrease in ratio and to increase in the prevalence of values below 1.0 were found in more hypertriglyceridemic patients. Finally, the ratio showed significant lower values (p <0.05) in 2-and 3-vessel disease groups compared to those obtained in l-vessel group (1.16+0.24 in the single group. 1.05-t:0.22 in the 2-vessel group and 1.03+0.21 in the 3-vessel group). Moreover. there was a significant inverse association between the ratio and triglyceride level (p<0.0001) and a significant positive association with HDI< cholesterol level ~ < 0 . 0 0 1 ) in all groups. These results suggest that LDL cholesterol:plasma apoB ratio is strongly associated with triglyceride and HDL: cholesterol in CAD-patients. and seems to be a better discri minstor for the extent of coronary artery disease angiographically defined.
A new direct LDLC method, based on immunoseparation (IS) has recently been reported (Sigma Diagnostics) and we have evaluated this along with another technique based on selective LDLC precipitation (P) (Immuno Ltd), in particular examining the effects of high triglyceride and nonfasting samples when the Friedwald formula (Ff) becomes invalid. Correlations with calculated LDLC and apoB (lncstar) and stability studies were also performed. All cholesterols and triglycerides (TG) were performed on an Ektachem 250 and ApoB on a Cobas Fara. 22 pairs of fasting and nonfasting samples were obtained and TG in the form of intralipid (Clintec) dilutions was added to 25 fasting sera to increase concentrations by up to 30 mM (2700 mg/dl). 3 sera were analysed each day after storage at 2-8 and -20 degrees C for 10 days. Fasting LDLC was 3.92 (SD 1.20) by IS and 5.27 (1.16) by P, nonfasting 3.91 (1.[6) and 5.05 (1.21). No significant trends in LDLC were observed with either method with the addition of increasing TG concentrations and samples appeared stable at both temperatures for up to 10 days. Correlation coefficient (n=20) between LDLC by IS and P was 0.89, between IS and calculated 0.80, P and calculated 0.71, and between apoB and IS 0.77, ApoB and P 0.55 and Apo B and calculated 0.70. Both direct LDLC methods appear promising, particularly where Ff cannot be used with nonfasting samples and when TG is > 4 . 5 mM(170 mg/dl).
462
464
HYDROPHOBIC INTERACTIONS IN PLASMA PROTEINS AND PATHOGENESIS OF ATHEROSCLEROSIS B. Lipinski H.S. Research Laboratory, Cambridge MA 02139, U.S.A
A NEW METHOD FOR THE RAPID SEPARATION OF PLASMA LIPOPROTEINS J.M. Graham, J.A. Higgins, T. Gillot School of Biomolecular Sciences, Liverpool John Moores University, Liverpool L3 3AF, UK; Dept of Molecular Biology, University of Sheffield, Sheffield SI0 2TN, UK; Northern General Hospital, Sheffield $5 7AU,UK.
Native plasma proteins are characterised by their relative hydrophilicity which prevents undesirable protein-protein and protein-cell interactions. Hydrophobic regions of proteins are normally buried in the interior of proteins and are stabilised by the intramolecular disulfide (S-S) bonds. These regions can be exposed in proteins by limited proteolysis and/or unfolding of the polypeptide chains resulting in a S-S exchange from intra- to intermolecular bonds. Limited reduction of human serum albumin (HSA) (e.g. with ascorbic acid) resulted in the formation of soluble hydrophobic multimers. A similar hydrophobic macromolecular protein complex (MPC) was identified in human plasma and was shown to be significantly increased in subjects with atherosclerosis. A characteristic feature of MPC is its ability to become incorporated into the fibrin clot during clotting of fibrinogen with thrombin. Fibrin clots obtained from the patient with atherosclerosis contained not only 50-80 % of fibrinogen-unrelated protein, but were also completely resistant to fibrinolysis induced with plasminogen activators. Incorporation of MPC into fibrin clots can be inhibited by certain amphiphilic drugs such as aurin tricarboxylic acid and suramin by virtue of the interaction of their hydrophobic moieties with those of fibrin monomers. Increased concentration of MPC in blood may thus contribute to pathogenesis of atherosclerosis making fibrin deposits refractory to fibrinolysis.
Variations in the relative amounts of plasma lipoprotein classes and/or molecular changes in their lipid components and apoproteins, are important factors in the development of atherosclerosis, A new equilibrium centrifugation method, which avoids high salt gradients, provides high resolution and concentration of lipoproteins for electrophoretic, compositional and structural analysis. The method uses a new non-ionic medium, Iodixanol, available as a 60% (w/v) solution called OptiPrep (Nycomed Pharma AS, Oslo). Plasma is adjusted to 12.5% lodixanol; overlaid with 0.1-0.2x its volume of saline and centrifuged at approx 350,000g,v for 2.5-3 h in a vertical or near-vertical rotor. Using 3.9 ml tubes, unloaded dense-end first; soluble proteins are contained in the first 1.0 ml, HDL in the 1.2 - 2.4 ml fraction; LDL in the 2.6 - 3.4 ml fraction and VLDL in the 3.6 - 3.9 ml fraction. There is little overlap between the three classes. Fractions can be analysed directly, by gel electrophoresis and for lipid content. The method may resolve different sub-classes of these lipoproteins; this is currently being investigated. Because of the concentration effect achieved by the gradient, Lp(a), which is recovered in a single 0.25 ml fraction, can be easily demonstrated on gels when levels in the whole plasma are below the limits of detection. The technique thus provides a means of fractionating and concentrating all plasma lipoproteins in a single step: it should prove to be a valuable diagnostic and analytical tool in studies of lipoprotein metabolism.