Linnéa Sjöblom

Determination of HDL2 cholesterol by precipitation with dextran sulfate and magnesium chloride: Establishing optimal conditions for rat plasma

Optimal conditions for analyzing HDL2 cholesterol in small amounts of rat plasma have been studied using different concentrations of dextran sulfate and MgCl2 to precipitate lipoproteins containing apolipoprotein B and/or apo E. When the MgCl2 level was 91 mM, the supernate cholesterol was rather constant at a level of about 50–60% of the total plasma cholesterol concentration. Immunochemical determination of the apo A-I content indicated that no major losses of the HDL2 fraction took place under these conditions. The recovery of about 96% of HDL2 lipoproteins after the precipitation of rat plasma and the almost complete absence of lipoproteins belonging to the VLDL, LDL and HDL1 fractions was demonstrated by agarose gel electrophoresis. Thus, the method should be suitable for screening the HDL2 cholesterol content in small volumes of rat plasma.

Effect of dietary proteins on hepatic and plasma lipids, and some properties of major plasma lipoprotein fractions during dietary-induced hypercholesterolemia in male rats

In order to characterize further the cholesterolemic effect of casein compared with soybean protein isolate, each of these proteins were fed to male rats at different levels of dietary cholesterol administration. An increase in the dietary cholesterol level from zero to 0.25% led to a several-fold increase in plasma triacylglycerols with a comparatively small rise in plasma cholesterol. Further increase in the cholesterol content of diet to 0.5, 1 and 2% resulted in a return of plasma triacylglycerols to normal or even subnormal values, whereas the plasma cholesterol values rose progressively. The triacylglycerolemic and cholesterolemic effects were 2-3-fold higher with casein diet than with soybean protein diet, respectively. A major part of the increased plasma cholesterol value was accounted for by cholesterol in the very-low-density lipoprotein (VLDL) fraction. Even at moderate levels (0.05-0.5%), the dietary cholesterol administration produced a series of changes in the electrophoretic pattern of plasma lipoproteins. These changes were more prominent in the rats fed casein diet, which often showed an enhanced VLDL band even on a cholesterol-free diet. An additional lipoprotein band, localized between VLDL and the LDL/HDL1 band, was observed in several rats fed casein diet with 0.05 or 0.1% cholesterol, and in rats fed soybean protein diet containing 0.25 or 0.5% cholesterol. In contrast, at the 0.25 and 0.5% levels of dietary cholesterol, plasma of casein-fed rats contained only one broad band within the VLDL-LDL/HDL1 region. All these findings indicate that casein diet promotes the appearance of one or more specific type(s) of cholesterol-induced plasma lipoprotein particles even at a comparatively low level of dietary cholesterol.

Dietary protein and fatty acid composition of liver lipids in the rat

In order to compare the effects of different sources of dietary protein on the fatty acid composition of phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylinositols (PI), cholesteryl esters and triacylglycerols, male rats were fed for a 4-week period on cholesterol-free, or cholesterol-containing, diets based on casein, or soybean protein and olive oil. The most conspicuous difference observed was the occurrence of significantly higher levels of 5,8,11-eicosatrienoic acid, 20:3 (n - 9), in the different lipid classes of casein-fed, compared with soybean protein-fed, animals. In the PI fraction of livers from the groups of rats fed casein diet, this fatty acid amounted to between 9.9 and 13.3% by weight of the total fatty acids. Phospholipids from livers of casein-fed rats contained increased levels of oleic acid, 18:1 (n - 9) (in PC and PE) and reduced levels of stearic acid (18:0). Moreover, in this group of rats PI contained a reduced level of arachidonic acid, 20:4 (n - 6). A casein-related decrease in the linoleic acid, 18:2 (n - 6), content of PC and PE was observed only in the rats fed on cholesterol-free diet. Effects on the fatty acid composition were also observed in the triacyglycerol and cholesteryl ester fractions, in which the rats fed casein diet showed higher levels of palmitoleic acid, 16:1 (n - 7) (cholesterol-supplemented diet) and lower values for linoleic acid, than the soybean protein-fed rats.

Improved banding pattern of rat plasma lipoproteins developed by agarose gel electrophoresis at pH 7.0

The use of a conventional agarose gel electrophoretic method to separate rat plasma lipoproteins resulted in a rather poor resolution of lipoproteins of lower density. Therefore, attempts were made to improve the resolution by running the electrophoresis under different conditions. It was shown that rat plasma lipoproteins could be separated into at least three fractions by agarose gel electrophoresis in phosphate buffer at pH 7.0. Using rat plasma lipoproteins isolated by sequential flotation as standards, these fractions were shown to correspond to high-density lipoprotein (HDL2), very-low-density lipoprotein (VLDL) and a mixed low-density lipoprotein (LDL)/high-density lipoprotein (HDL1) fraction. Since VLDL is completely separated from the other lipoprotein classes the method could be used to monitor changes in plasma VLDL.

Effect of metoprolol on plasma lipids and arterial intimal lipid deposition in spontaneously hypertensive rats

The purpose of the present study was to characterize possible effects of dietary-induced plasma lipid elevations on the development of arterial lesions in spontaneously hypertensive rats (SHR) and to reveal any influence of treatment with metoprolol on these parameters. Metoprolol treatment caused an 8% decrease in heart rate and a 13% decrease in blood pressure and led to a rise in plasma triglycerides, 24%, 17% and 34% after 1, 3 and 6 months of metoprolol treatment, respectively. However, no effect on plasma triglycerides was observed after 9 months of metoprolol treatment while a reduced cholesterolemic response was observed. Intimal proliferations containing accumulations of lipids were observed in small intramural branches of coronary arteries (greater than 100 microns) in 11 of 31 control rats fed the atherogenic diet for 9 months. In contrast, similar changes were observed in only 1 of 34 metoprolol-treated rats fed an otherwise identical diet. The corresponding figures for the frequency of lipid containing intimal plaques in aorta were 6/19 in controls and 2/24 in the metoprolol-treated group.

Accumulation of cholesteryl esters and triglycerides in cultured macrophages incubated with plasma very low density lipoproteins from rats fed on casein or soybean protein diets containing moderate levels of cholesterol

Even when administered at a comparatively low level, dietary cholesterol produces significant changes in the properties of plasma lipoproteins in rats, particularly the d less than or equal to 1.006 g/ml fraction (VLDL). The occurrence of these changes is promoted by dietary casein. To test the hypothesis that these dietary-induced perturbations might include properties influencing lipoprotein-cell interactions of relevance to atherogenesis, cultured mouse peritoneal macrophages were incubated with VLDL isolated from male rats fed on diets containing either 0, 0.25 or 0.5% cholesterol with casein or soybean protein, respectively, as the sole source of protein. No increase in cholesteryl ester content, and a comparatively small rise in triglyceride content, was observed in macrophages incubated with VLDL from rats fed on cholesterol-free diets. In contrast, a significant and apparently saturable cellular accumulation of cholesteryl esters as well as triglycerides was produced by VLDL from cholesterol-fed rats. The curves showing cellular lipid accumulation versus VLDL-protein (or VLDL-cholesterol) content in the cell medium indicated different cellular affinity for VLDL from casein-fed rats in comparison with VLDL from soybean protein-fed rats. The apoprotein composition of VLDL differed between groups of rats fed on different types of dietary protein with higher proportions of apo Cs in the casein-fed rats. In addition, cholesterol feeding resulted in increased proportions of apo A-I and apo A-IV in the plasma VLDL fraction.

Comparison of Dietary Casein and Soybean Protein Effects on Plasma Lipid and Gastrin Levels, Hepatic Δ6-Desaturase Activity and Coronary Arteriosclerosis in Male Sprague-Dawley Rats

An increased concentration of gastrin was observed in plasma of male Sprague-Dawley strain rats fed on soybean protein diet for a 9-month period, compared with rats fed on casein diet. Both diets contained 0.5% (w/w) cholesterol. Protein-dependent differences were also observed in the fatty acid pattern of hepatic phospholipids, hepatic Δ6-desaturase activity, and plasma cholesterol. No signs of arteriosclerosis were observed in the aortas. Sixty percent of the hearts showed various degrees of lipid staining in coronary arterial branches of different sizes. Despite a large difference in plasma cholesterol level, there was no quantitative or qualitative difference between groups in the occurrence of coronary lipid staining.

Effects of diet and metoprolol on lipid levels in the blood plasma and morphology of the heart and intramural branches of coronary arteries of spontaneously hypertensive male rats. A 9-month study.

Semipurified diets containing 0.5% cholesterol were used in a 9-month study with spontaneously hypertensive male rats to characterize the effects of the protein source (casein vs. soybean protein), and the selective beta 1-adrenoceptor antagonist metoprolol on both lipid levels in blood plasma and the aorta, and on the morphology of intramural branches of coronary arteries. Raised blood lipid levels were observed in these rats. A significant decline in HDL2 cholesterol took place, while plasma cholesterol belonging to lipoprotein fractions of lower density increased. Metoprolol treatment led to a substantial elevation of the plasma triacylglycerol level and, with time, a reduced cholesterolemic response. The use of soybean protein instead of casein had a persistent plasma lipid-lowering effect. Arteriosclerotic changes in the form of musculo-elastic thickenings, intimal cushions and homogeneous hyalin deposits appeared in the intramural coronary arteries of rats in all groups after 9 months on the diet. However, intimal deposition of lipid was only present in rats belonging to the casein group not treated with metoprolol. Rats of this group also showed more severe myocardial lesions in the form of scar tissue with or without inflammatory cell reaction.