Detection of low density lipoprotein particle fusion by proton nuclear magnetic resonance spectroscopy.

Recent evidence suggests that fusion of low density lipoprotein (LDL) particles is a key process in the initial accumulation of lipid in the arterial intima. In order to gain a better understanding of this early event in the development of atherosclerosis, it would thus be necessary to characterize the process of LDL fusion in detail. Such studies, however, pose severe methodological difficulties, such as differentiation of particle fusion from aggregation. In this paper we describe the use of novel methodology, based on 1H NMR spectroscopy, to study lipoprotein particle fusion. To test the methodology, we chose proteolytic fusion of LDL particles, an in vitro model that has been well characterized in our laboratory. The spectroscopic data suggested that proteolysis of LDL with alpha-chymotrypsin induced slow initiation of fusion, which was followed by particle fusion at an increased rate. Moreover, 1H NMR spectroscopic data on different kinds of LDL interactions, for example, when LDL formed aggregates with antibodies against human apolipoprotein B-100, were obtained and compared with the electron microscopic characteristics of these preparations. An important finding was that limited aggregation of LDL particles did not disturb the 1H NMR spectroscopic parameters used for the detection of particle fusion and preserved the physico-chemical information on the particles. The 1H NMR methodology developed is sensitive to and specific for low density lipoprotein (LDL) fusion and may also allow for studies of the fate of LDL particles in other in vitro preparations that mimic the arterial interactions in vivo.

[1]  M Ala-Korpela,et al.  Assessment of cholesteryl ester transfer protein function in lipoprotein mixtures by 1H NMR spectroscopy , 1997, NMR in biomedicine.

[2]  V. Tertov,et al.  Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome. , 1992, Journal of Biological Chemistry.

[3]  P. Kovanen,et al.  Aggregation and fusion of modified low density lipoprotein. , 1996, Journal of lipid research.

[4]  M. Spraul,et al.  750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. , 1995, Analytical chemistry.

[5]  R. Krauss,et al.  Development of a proton nuclear magnetic resonance spectroscopic method for determining plasma lipoprotein concentrations and subspecies distributions from a single, rapid measurement. , 1992, Clinical chemistry.

[6]  M. Ala-Korpela,et al.  Evidence for distinct behaviour of phosphatidylcholine and sphingomyelin at the low density lipoprotein surface. , 1997, Biochemical and biophysical research communications.

[7]  G. Bondjers,et al.  Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells. , 1993, The Journal of biological chemistry.

[8]  M Ala-Korpela,et al.  1H NMR-based absolute quantitation of human lipoproteins and their lipid contents directly from plasma. , 1994, Journal of lipid research.

[9]  P. Kovanen,et al.  Fusion of proteolyzed low-density lipoprotein in the fluid phase: a novel mechanism generating atherogenic lipoprotein particles. , 1995, Biochemistry.

[10]  J. Guyton,et al.  Development of the lipid-rich core in human atherosclerosis. , 1996, Arteriosclerosis, thrombosis, and vascular biology.

[11]  H. Hauser,et al.  Low density lipoprotein particle size and core cholesteryl ester physical state affect the proton NMR magnetic environment of fatty acid methylene and methyl nuclei. , 1996, Journal of lipid research.

[12]  M. Mims,et al.  Altered ultrastructural morphology of self-aggregated low density lipoproteins: coalescence of lipid domains forming droplets and vesicles. , 1991, Journal of lipid research.

[13]  K. Williams,et al.  The response-to-retention hypothesis of early atherogenesis. , 1995, Arteriosclerosis, thrombosis, and vascular biology.

[14]  A. Annila,et al.  Proteolysis and Fusion of Low Density Lipoprotein Particles Strengthen Their Binding to Human Aortic Proteoglycans (*) , 1995, The Journal of Biological Chemistry.

[15]  P. Sadler,et al.  Copper-induced LDL peroxidation investigated by 1H-NMR spectroscopy. , 1995, Biochimica et biophysica acta.

[16]  A. J. Shaka,et al.  Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients , 1995 .

[17]  H. Kruth.  The fate of lipoprotein cholesterol entering the arterial wall , 1997, Current opinion in lipidology.

[18]  P. Kovanen,et al.  Proteolysis and fusion of low density lipoprotein particles independently strengthen their binding to exocytosed mast cell granules. , 1994, The Journal of biological chemistry.

[19]  R. Havel,et al.  The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. , 1955, The Journal of clinical investigation.

[20]  D. Steinberg,et al.  Enhanced Macrophage Uptake of Low Density Lipoprotein after Self‐Aggregation , 1988, Arteriosclerosis.

[21]  I. Tabas,et al.  Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages. , 1991, The Journal of biological chemistry.

[22]  G. Pasquinelli,et al.  Electron microscopy of lipid deposits in human atherosclerosis. , 1989, Scanning microscopy.

[23]  Rudolph Willem,et al.  A computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints: Analysis of overlapping 13C‐2H multiplets of 13C enriched metabolites from cell suspensions incubated in deuterated media , 1996, Magnetic resonance in medicine.

[24]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[25]  N. Simionescu,et al.  Intimal thickenings of human aorta contain modified reassembled lipoproteins. , 1995, Atherosclerosis.

[26]  Mika Ala-Korpela,et al.  1H NMR spectroscopy of human blood plasma , 1995 .

[27]  Jimmy D Bell,et al.  Effects of n-3 fatty acids on the NMR profile of plasma lipoproteins. , 1996, Journal of lipid research.

[28]  Lounila,et al.  Effects of orientational order and particle size on the NMR line positions of lipoproteins. , 1994, Physical review letters.

[29]  J. Guyton,et al.  Ultrastructure of the human aortic fibrolipid lesion. Formation of the atherosclerotic lipid-rich core. , 1986, The American journal of pathology.

[30]  P. Kovanen,et al.  Proteolytic enzymes of mast cell granules degrade low density lipoproteins and promote their granule-mediated uptake by macrophages in vitro. , 1989, The Journal of biological chemistry.

[31]  N. Simionescu,et al.  In vitro formation of oxidatively-modified and reassembled human low-density lipoproteins: antioxidant effect of albumin. , 1993, Biochimica et biophysica acta.

[32]  M. Jauhiainen,et al.  Human Arterial Proteoglycans Increase the Rate of Proteolytic Fusion of Low Density Lipoprotein Particles* , 1997, The Journal of Biological Chemistry.

[33]  K. Williams,et al.  Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins. , 1996, The Journal of clinical investigation.

[34]  Jimmy D Bell,et al.  1H NMR studies of human blood plasma Assignment of resonances for lipoproteins , 1987, FEBS letters.

[35]  Y. Hiltunen,et al.  A lineshape fitting model for 1H NMR spectra of human blood plasma , 1991, Magnetic resonance in medicine.

[36]  A. Fogelman,et al.  Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. A deep-etch and immunolocalization study of ultrarapidly frozen tissue. , 1991, Arteriosclerosis and thrombosis : a journal of vascular biology.