Alex V. Nichols

Characterization of human high-density lipoproteins by gradient gel electrophoresis

Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.

Lipoproteins in atherosclerosis

Abstract 1.1. Serum lipoprotein patterns as studied ultracentrifugally in the human indicate lipoprotein transport may be interpreted as reflecting varying degrees of a lipid metabolic error. 2.2. The nature of several lipid transport disturbances have been described for a variety of experimental procedures in the rabbit. In all disturbances there is a strong positive correlation between elevation of the S f 10–30 class of lipoproteins and the rate of development of atherosclerosis. Lipoproteins of the classes of S f 10 and less and S f 40–50 and higher do not show this correlation. Total serum cholesterol is positively related to the development of atherosclerosis only in those experimental procedures which allow for the increased serum cholesterol to be largely in the S f 10–30 class. 3.3. A critical comparison has been made, in which the analysis of total cholesterol and S f 12–20 lipoproteins was done on aliquots of the same serum sample, of both levels in normals vs. atherosclerotics, using myocardial infarctions as the test group. The over-all correlation of S f 12–20 lipoprotein levels with atherosclerosis is two to four times as great as that for serum cholesterol levels with atherosclerosis. 4.4. The S f 12–20 lipoprotein levels are associated with atherosclerosis, independently of their relationship with serum cholesterol. The S f 12–20 levels account for the bulk of the over-all predictive segregation of atherosclerotics from normals. 5.5. The serum cholesterol level is very much less, if at all, associated with atherosclerosis, when considered independently of its association with the S f 12–20 levels. 6.6. One-year follow-up studies of patients with coronary artery disease indicate that early recurrence of myocardial infarction is significantly associated with elevated S f 12–20 lipoprotein levels. An approximate estimate of recurrence rate as a function of S f 12–20 level may be made from the data on thirty-nine recurrences in the follow-up study. At 50 mg. per cent S f 12–20 the chance of recurrence is ~6 per cent in one year; at 110 mg. per cent the chance is ~18 per cent. 7.7. In acute myocardial infarction the prognosis for survival is worsened with highly elevated S f 12–20 lipoprotein levels, as determined during the acute phase. 8.8. Follow-up studies show that the reduction of S f 12–20 lipoprotein levels by dietary restriction of fats and cholesterol gives a significant degree of protection against recurrent myocardial infarction in patients with coronary artery disease whose levels before dietary restriction ranged above 80 mg. per cent. 9.9. The only pharmacologic agent which rapidly shifts the lipoprotein pattern in humans in the direction of normality is parenteral heparin. The possibility is considered that a deficiency of heparin or a heparin-like substance may be involved in causing the basic lipid metabolic defect in humans.

Particle distribution of human serum high density lipoproteins.

Density gradient ultracentrifugation of human serum high density lipoproteins (HDL) from both normolipemic males and females results in a distribution of HDL concentration versus subfraction hydrated density which has three maxima. Gradient gel electrophoresis of total HDL is characterized by three banding maxima, the positions of which suggest the presence of three particle size ranges: I. 10.8-12.0 nm, II. 9.7-10.7 nm, and III. 8.5-9.6 nm. Gradient gel electrophoresis of density gradient subfractions established an inverse relationship between particle size and particle hydrated density which was corroborated by electron microscopy and analytic ultracentrifugation. Comparison of male HDL from size ranges I, II, and III with female HDL from the same size ranges showed only small differences in the mean value of the peak F degrees 1.20 rate, size, molecular weight, protein weight percent, and weight protein/weight phospholipid. Major differences between males and females were seen in the relative amounts of HDL in density gradient subfractions 1-3 (size range I material) and 11-12 (size range III material); the percent total HDL in the group of subfractions 1-3 was greatly increased in female HDL while that of the group of subfractions 11-12 was increased in the male HDL. These studies indicate the presence of at least three major components in HDL instead of two (HDL2 and HDL3) and that peak F degrees 1.20 rate differences in HDL schlieren patterns between males and females are a function of the relative levels of these three components.

Electron microscopy of human serum lipoproteins using negative staining

Abstract Human serum lipoproteins were separated by ultracentrifugation into 3 fractions: high density lipoproteins, low density lipoproteins, and very low density lipoproteins including chylomicrons. The structures of the various fractions were studied with the electron microscope after negative staining with sodium phosphotungstate. The high density lipoproteins appear to be structures 86 A in average diameter possessing an electron-dense central region. The lipoproteins are composed of subunits which in some cases appear to be elongated with an estimated short axis of 36 A and long axis of 64 A. The high density lipoproteins may further be fractionated into their subclasses, HDL2 and HDL3. The HDL3 molecules are considerably smaller in average diameter (65 A) than the HDL2 (95 A). The low density lipoproteins appear spherical with a mean diameter of 216 A and they display a high degree of deformability, but they have no visible subunit structures. Bridge-like connections between adjacent lipoproteins also appear to be present. The very low density lipoproteins plus chylomicrons are extremely heterogeneous in size and shape and range from 230–5000 A in diameter. The smaller molecules seem to be spherical while the extremely large ones are flattened and irregular in shape. There is no apparent substructure either within or at the periphery of these particles.

Electron microscopic study on reassembly of plasma high density apoprotein with various lipids.

Products resulting from the sonification of mixtures of plasma high density lipoprotein apoprotein and specific lipids were studied by electron microscopy using negative staining. Sonicates of apoprotein plus lecithin produced disc-shaped structures which stacked in aggregates with a 50–55-A repeat; the discs were 100–200 A in diameter. Incorporation of unesterified cholesterol into the mixture produced structures morphologically similar to those observed in sonicates of apoprotein plus lecithin. Disc-shaped particles from sonified mixtures of apoprotein, lecithin and unesterified cholesterol were ultracentrifugally isolated in the d 1.063–1.21 g/ml fraction and were incubated with a plasma d > 1.21 g/ml fraction containing lecithin: cholesterol acyltransferase activity. Electron microscopy following the incubation procedure showed a transformation of the disc-like structures into approximately spherical particles (50–100 A diameter). Similar spherical particles were also obtained after sonification of apoprotein-lecithin-unesterified cholesterol-cholesteryl ester mixtures. Results indicate a requirement for the presence of cholesteryl esters to maintain normal morphology of plasma high density lipoproteins.

EVIDENCE FOR LIPOLYTIC ACTION BY HUMAN PLASMA OBTAINED AFTER INTRAVENOUS ADMINISTRATION OF HEPARIN

Summary 1. Plasma from human subjects who have received intravenous heparin a short time previously, when incubated with certain lipoproteins for period of 4 to 8 hours at 37°C, has been found to cause partial hydrolysis of the glyceride component of the lipoprotein with concomitant release of fatty acids. Sera from the same individuals before heparin administration did not cause measurably real hydrolysis under the same conditions. 2. Heating the plasma before incubation prevents the reaction, but of a number of common enzyme inhibitors tried, only sodium arsenite is effective. 3. Our results are consistent with—and may at least partially explain—certain other lipoprotein changes which may be classed as heparin effects, e.g., the observed redistribution of lipoproteins shown by the ultracentrifuge, and the cleaning of turbidity in egg lipoprotein solutions.

Plasma Lipoproteins in Familial Lecithin:Cholesterol Acyltransferase Deficiency: Effects of Incubation with Lecithin: Cholesterol Acyltransferase in vitro

To study the effect of lecithin: cholesterol acyltransferase (LCAT) on the plasma lipoproteins of patients with familial LCAT deficiency, whole plasma or the lipoprotein fraction of d smaller than 1.006 g/ml (VLDL) was incubated in the presence of LCAT and subsequently examined by chemical, physical, and immunological techniques. The following occured upon incubating either hyperlipemic or nonlipemic plasma: The concentrations of polar lipids decreased, particulary in the large molecular weight lipoprotein subfraction of d 1.019-1.063 g/ml (LDL2) and in the lipoprotein fraction of 1.06301.25 g/ml (HDL). The concentration of cholesteryl ester (CE) increased, particularly in the VLDL and in the lipoprotein fractions of d 1.006-1.019 g/ml (LDL1) and LDL2. The concentration of arginine-rich apolipoprotein decreased in the HDL and increased in the VLDL and LDL1. The concentrations of the C-apoliproteins appeared to change in the opposite direction. The concentration of apolipoprotein B in the LDL increased concomitantly with an increase in the concentration and flotation rsate of the small LDL2. The concentration apolipoprotein A-I in the HDL increased; and a major component in the HDL fraction became identical in apperance to normal HDL. Upon incubating a patients isolated VLDL in the presence of LCAT, lipoproteins with properties similar to normal LDL2 were formed. These experiments show that the LCAT reaction can alter the apolipoprotein content and physical properties as well as the lipid content of the patients lipoproteins.

Plasma lipoproteins in familial lecithin: cholesterol acyltransferase deficiency: physical and chemical studies of low and high density lipoproteins

LOW DENSITY LIPOPROTEINS (LDL) AND HIGH DENSITY LIPOPROTEINS (HDL) FROM THE PLASMA OF PATIENTS WITH FAMILIAL LECITHIN: cholesterol acyltransferase (LCAT) deficiency have been characterized by gel filtration, analytical ultracentrifugation, and gel electrophoresis, and their relative content of lipid and protein has been determined. The LDL of d 1.019-1.063 g/ml show marked heterogeneity. A subfraction of the LDL emerges from columns of 2% agarose gel with the void volume, has corrected flotation rates (S(f) degrees ) in the range of 20-400, and contains 4-10 times as much unesterified cholesterol, phosphatidylcholine, and triglyceride per mg protein as normal LDL. A major subfraction of the LDL emerges from the gel in the same general position as normal LDL, but exhibits somewhat higher flotation rates and contains 1.5-3 times as much unesterified cholesterol and phosphatidylcholine and 13 times as much triglyceride per mg protein. The HDL, shown to be heterogeneous in earlier studies, are mainly comprised of molecules which have flotation rates of F(1.20) 3-20, migrate in the alpha(1)-alpha(2) region on electrophoresis, and contain about 12 times as much unesterified cholesterol and 5 times as much phosphatidylcholine per mg protein as normal HDL. Smaller molecules are also detected, which have flotation rates of F(1.20) 0-3, migrate in the prealbumin region on electrophoresis, and contain only slightly more unesterified cholesterol and phosphatidylcholine per mg protein than normal HDL.

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin: Cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.