Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

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Nile red: a selective fluorescent stain for intracellular lipid droplets.

Apolipoprotein E (apoE) is a ligand for receptors that clear remnants of chylomicrons and very low density lipoproteins Lack of apoE is, therefore, expected to cause accumulation in plasma of cholesterol-rich remnants whose prolonged circulation should be atherogenic ApoE-deficient mice generated by gene targeting were used to test this hypothesis and to make a mouse model for spontaneous atherosclerosis The mutant mice had five times normal plasma cholesterol, and developed foam cell-rich depositions in their proximal aortas by age 3 months These spontaneous lesions progressed and caused severe occlusion of the coronary artery ostium by 8 months The severe yet viable phenotype of the mutants should make them valuable for investigating genetic and environmental factors that modify the atherogenic process

https://science.sciencemag.org/content/sci/258/5081/468.full.pdf

Spontaneous Hypercholesterolemia and Arterial Lesions in Mice Lacking Apolipoprotein E

The amount of abdominal visceral adipose tissue measured by computed tomography is a critical correlate of the potentially "atherogenic" metabolic disturbances associated with abdominal obesity. In this study conducted in samples of 81 men and 70 women, data are presented on the anthropometric correlates of abdominal visceral adipose tissue accumulation and related cardiovascular disease risk factors (triglyceride and high-density lipoprotein cholesterol levels, fasting and postglucose insulin and glucose levels). Results indicate that the waist circumference and the abdominal sagittal diameter are better correlates of abdominal visceral adipose tissue accumulation than the commonly used waist-to-hip ratio (WHR). In women, the waist circumference and the abdominal sagittal diameter also appeared more closely related to the metabolic variables than the WHR. When the samples were divided into quintiles of waist circumference, WHR or abdominal sagittal diameter, it was noted that increasing values of waist circumference and abdominal sagittal diameter were more consistently associated with increases in fasting and postglucose insulin levels than increasing values of WHR, especially in women. These findings suggest that the waist circumference or the abdominal sagittal diameter, rather than the WHR, should be used as indexes of abdominal visceral adipose tissue deposition and in the assessment of cardiovascular risk. It is suggested from these data that waist circumference values above approximately 100 cm, or abdominal sagittal diameter values > 25 cm are most likely to be associated with potentially "atherogenic" metabolic disturbances.

Waist circumference and abdominal sagittal diameter: Best simple anthropometric indexes of abdominal visceral adipose tissue accumulation and related cardiovascular risk in men and women

Three lines of evidence are presented that low density lipoproteins gently extracted from human and rabbit atherosclerotic lesions (lesion LDL) greatly resembles LDL that has been oxidatively modified in vitro. First, lesion LDL showed many of the physical and chemical properties of oxidized LDL, properties that differ from those of plasma LDL: higher electrophoretic mobility, a higher density, higher free cholesterol content, and a higher proportion of sphingomyelin and lysophosphatidylcholine in the phospholipid fraction. A number of lower molecular weight fragments of apo B were found in lesion LDL, similar to in vitro oxidized LDL. Second, both the intact apo B and some of the apo B fragments of lesion LDL reacted in Western blots with antisera that recognize malondialdehyde-conjugated lysine and 4-hydroxynonenal lysine adducts, both of which are found in oxidized LDL; plasma LDL and LDL from normal human intima showed no such reactivity. Third, lesion LDL shared biological properties with oxidized LDL: compared with plasma LDL, lesion LDL produced much greater stimulation of cholesterol esterification and was degraded more rapidly by macrophages. Degradation of radiolabeled lesion LDL was competitively inhibited by unlabeled lesion LDL, by LDL oxidized with copper, by polyinosinic acid and by malondialdehyde-LDL, but not by native LDL, indicating uptake by the scavenger receptor(s). Finally, lesion LDL (but not normal intimal LDL or plasma LDL) was chemotactic for monocytes, as is oxidized LDL. These studies provide strong evidence that atherosclerotic lesions, both in man and in rabbit, contain oxidatively modified LDL.

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Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.

In the past few years several methods have been developed for the analysis of serum lipoproteins Lindgren, Elliott, and Gofman (1) have utilized the relatively low density of the lipoproteins to separate them from the other serum proteins by ultracentrifugal flotation Quantitation was subsequently performed by refractometric methods in the analytical ultracentrifuge Separations of lipoproteins have also been made by Cohn fractionation in cold ethanol, and the quantities of lipoprotein have been estimated from the lipid content of the fractions (2, 3) Widely used at the present time is the method of zone electrophoresis with quantitation either by staining (4) or by chemical analysis of eluates from the support

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The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum

Colorimetric determination of blood lipides.

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Tissue distribution of c14 after the intravenous injection of labeled chylomicrons and unesterified fatty acids in the rat

In Vivo Effect of Anti-Heparin Agents on Serum Lipids and Lipoproteins

Fasting in the rat is accompanied by an increase in serum cholesterol concentration reflecting an increase in high-density lipoproteins. The feeding of carbohydrate results in decreases in both low- and high-density lipoproteins, the former occurring acutely, and the latter occurring more slowly. In the fasting rat the injection of protamine, an antiheparin agent, produces an increase in all serum lipids, but the increase occurs in the low-density lipoproteins. In the rat fed carbohydrate this lipemia-inducing effect of protamine is practically abolished. The feeding of carbohydrate has no effect, however, on the rate of clearance of intravenously administered chylomicrons. These phenomena are discussed in relation to current theories of lipid transport.

Joseph H. Bragdon

Papers

The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum

Colorimetric determination of blood lipides.

Tissue distribution of c14 after the intravenous injection of labeled chylomicrons and unesterified fatty acids in the rat

In Vivo Effect of Anti-Heparin Agents on Serum Lipids and Lipoproteins

Effects of carbohydrate feeding on serum lipids and lipoproteins in the rat.