Characterization of equine plasma lipoproteins after separation by density gradient

1. Plasma lipoproteins from six thoroughbred horses were separated by density gradient ultracentrifugation. For each sample, lipoprotein bands were visualized by means of a prestained plasma control and characterized by electrophoretic, chemical and morphological analysis. 2. Very low density lipoproteins (VLDL) were isolated at d less than 1.018 g/ml. 3. Two clearly resolved bands were detected in the low density lipoprotein fraction (LDL). The density limits were evaluated as follows: LDL1(1.028 less than d less than 1.045 g/ml) and LDL2(1.045 less than d less than 1.070 g/ml). Marked differences were observed in the chemical composition and particle size of LDL1 and LDL2 fractions. 4. High density lipoprotein fraction (HDL) was usually isolated as a single band, distributed over the range 1.075 less than d less than 1.180 g/ml. However, chemical composition and particle size revealed heterogeneity in HDL subfractions. 5. The density limit of LDL and HDL bands varied in each animal, indicating differences in equine lipoprotein distribution.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

The lipid composition of serum lipoproteins in the harbour seal, Phoca vitulina

The density profile of serum lipoproteins and their lipid composition was studied in 12 adult, female harbour seals. The animals were sampled after an approximate 20 hr fast. The density profile of lipoproteins showed that the harbour seals displayed a distinct VLDL (density less than 1.006 g/ml) and HDL band (density about 1.125 g/ml), but no clear LDL band. There was a rather diffuse population of lipoproteins in the density range of 1.019-1.100 g/ml. Mean serum total cholesterol concentration was 5.7 mmol/l; about 60% of this cholesterol was located in the HDL fraction (density greater than 1.063 g/ml). The fasted seals were found to carry 4% of serum total lipids in chylomicrons. These lipoproteins consisted of 51% of triaclyglycerols (on the basis of total chylomicron lipids). The LDL (defined as heparin-manganese precipitable lipoproteins in VLDL and chylomicron-deficient serum) contained 49% of cholesterol and 43% of phospholipids (on the basis of total LDL lipids). The HDL (defined as heparin-manganese soluble lipoproteins in VLDL and chylomicron-deficient serum) contained 36% of cholesterol and 58% of phospholipids (on the basis of total HDL lipids).     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

Lipid metabolism in cultured cells. XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic and tumor virus-transformed human fibroblasts.

Serum lipoproteins control cell cholesterol content by regulating its uptake, biosynthesis, and excretion. Monolayers of cultured fibroblasts were used to study interactions with human high density (HDL) and low density (LDL) lipoproteins doubly labeled with [(3)H]cholesterol and (125)I in the apoprotein moiety. In the binding assay for LDL, the absence of specific LDL receptors in type II hypercholesterolemic fibroblasts was confirmed, whereas monolayers of virus-transformed human lung fibroblasts (VA-4) exhibited LDL binding characteristics essentially the same as normal lung fibroblasts. In the studies of HDL binding, specific HDL binding sites were demonstrated in normal and virus-transformed fibroblasts. In addition, type II hypercholesterolemic cells, despite the loss of LDL receptors, retained normal HDL binding sites. No significant competition was displayed between the two lipoprotein classes for their respective binding sites over a 5-fold concentration range. In VA-4 cells, the amount of lipoprotein required to saturate half the receptor sites was 3.5 micro g/ml (9 x 10(-9) M) for LDL and 9.1 micro g/ml (9 x 10(-8) M) for HDL. Pronase treatment reduced LDL binding by more than half but had no effect on HDL binding. Chloroquine, a lysomal enzyme inhibitor, stimulated net LDL uptake 3.5-fold by increasing internalized LDL but had essentially no effect on HDL uptake. Further experiments were conducted using doubly labeled lipoproteins to characterize the interaction of LDL and HDL with cells. While the cholesterol and protein moieties of LDL were incorporated into cells at similar rates, the uptake of the cholesterol moiety of HDL was 5 to 10 times more rapid than that of the protein component. Furthermore, the apoprotein component of LDL is extensively degraded following exposure, whereas the apoprotein moiety of HDL retains its macromolecular chromatographic characteristics. These results indicate that HDL and LDL bind to cultured cells at separate sites and that further processing of the two lipoprotein classes appears to take place by fundamentally different mechanisms.-Wu, J-D., J. Butler, and J. M. Bailey. Lipid metabolism in cultured cells XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic, and tumor virus-transformed human fibroblasts.     

Hyperlipoproteinemia in Nagase analbuminemic rats: effects of pravastatin on plasma (apo)lipoproteins and lecithin:cholesterol acyltransferase activity.

The present study demonstrates very high levels of plasma lipids and high density lipoprotein (HDL) apolipoproteins (apoA-I and apoE) in female Nagase analbuminemic rats (NAR) fed a semi-synthetic diet in order to further increase the hyperlipidemia present in this strain. Plasma apoB-containing lipoproteins (very low, intermediate, and low density lipoproteins) were also elevated in NAR. Plasma cholesterol was mainly present in lipoprotein particles with a density between 1.02 and 1.12 g/ml. Separation of lipoprotein classes by gel filtration showed that the major cholesterol-carrying lipoprotein fractions in NAR plasma are apoE-rich HDL and apoA-I-rich HDL. The high HDL levels in NAR are explained, at least partly, by the two- to threefold elevated activity of plasma lecithin:cholesterol acyltransferase (LCAT). The lysophosphatidylcholine generated in the LCAT reaction, as well as plasma free fatty acids, are bound to lipoproteins in NAR plasma. A study was carried out to determine whether the elevated LDL and aopoE-rich HDL levels could be corrected by administration of the HMG-CoA reductase inhibitor pravastatin (at a dose of 1 mg/kg per day). Pravastatin treatment results in a 43% decrease in plasma triglycerides in NAR, but not in Sprague-Dawley (SDR) rats, and had no significant effect on plasma total cholesterol, phospholipids apolipoproteins A-I, A-IV, B, or E, as well as on plasma LCAT activity levels in NAR or SDR.     

Effect of dietary cholesterol level on the composition of thoracic duct lymph lipoproteins isolated from nonhuman primates

The effect of two different levels of dietary cholesterol (0.16 mg/Kcal and 0.79 mg/cal) on the composition of thoracic lymph duct lipoproteins was studied in two species of nonhuman primates, Ceropithecus aethiops (African green monkey) and Macaca fascicularis (cynomolgus monkey). Diet was infused intraduodenally at a constant rate to facilitate comparisons among animals. The higher level of dietary cholesterol resulted in an increase in the amount of cholesteryl ester in lymph chylomicrons and VLDL. Cholesteryl oleate was the predominant cholesteryl ester present in lymph d less than 1.006 g/ml lipoproteins and it was the predominant cholesteryl ester formed from exogenous radiolabeled cholesterol. The percentage of saturated and monounsaturated cholesteryl esters in lymph chylomicrons and VLDL significantly increased with the higher dietary cholesterol level. The apoprotein distribution of chylomicrons and VLDL was qualitatively similar during infusions of both diets. The apoprotein B of intestinal chylomicrons and VLDL, termed apoprotein B2, was qualitatively similar during low and high cholesterol diet infusion and was significantly smaller than that of plasma LDL apoB, termed apoprotein B1, as indicated by its electrophoretic mobility in SDS-polyacrylamide gels. The major phospholipid present in lymph chylomicrons and VLDL was phosphatidylcholine and the phospholipid composition of the particles was not affected by diet. Lymph d greater than 1.006 g/ml lipoproteins were separated and the cholesterol mass distribution among lipoprotein fractions was found to be similar during both diet infusions. With an increase in the level of dietary cholesterol, the percentage esterification of cholesterol mass and of exogenous cholesterol radioactivity increased in LDL and HDL from lymph. Lymph LDL and HDL contained less free and esterified cholesterol when their composition was compared to that for these lipoproteins in plasma. We conclude that the primary effect of increased dietary cholesterol level was to increase the cholesteryl ester content of all lymph lipoproteins; cholesterol distribution among lymph lipoproteins was unaffected.     

Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts

Rat fibroblasts degraded human low density lipoprotein (LDL) very slowly, one-tenth to one-fortieth the rates observed in human fibroblasts. In rat cells, human LDL caused only very small increases in cell cholesterol content and acylCoA:cholesterol acyltransferase (ACAT) activity and caused only small decreases in beta-hydroxy-beta-methylglutaryl CoA (HMG CoA) reductase activity; in human cells, however, human LDL induced very large changes in all three of these parameters, as expected. The binding of human LDL to rat fibroblasts was not reduced by previous incubation with human LDL or with 25-hydroxycholesterol. Thus, in rat fibroblasts there appear to be few, if any, regulated high-affinity receptors that recognize human LDL. Rat LDL fractions (d 1.02-1.05 g/ml), in contrast, were degraded more rapidly than human LDL by rat fibroblasts, caused a significant increase in cell cholesterol content, an increase in ACAT activity, and a significant decrease in HMG CoA reductase activity. Moreover, the degradation of this rat LDL fraction by rat fibroblasts as a function of concentration was biphasic, i.e., there appeared to be a high-affinity component of degradation. Thus, it appears that rat fibroblasts do have a receptor for homologous lipoproteins. However, because both apoprotein B and apoprotein E are present in these rat lipoprotein fractions, the observed effects may relate to recognition of either or both of these apoproteins. The metabolism and metabolic effects of the conventionally defined high density lipoprotein (HDL) fraction of the rat by rat or human fibroblasts resembled those of human LDL in human fibroblasts. It is suggested that rat HDL may, because of its apo E content and higher concentration in rat plasma relative to that of LDL, play an important role in cholesterol homeostasis in vivo.     

The effect of serum lipoproteins on cholesterol content and sterol exchange in cultured human endothelial cells.

Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum. Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.     

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.     

A density gradient study of the lipoprotein and apolipoprotein distribution in the chicken, Gallus domesticus

Plasma lipoproteins from 5-week old male chickens were separated over the density range 1.006-1.172 g/ml into 22 subfractions by isopycnic density gradient ultracentrifugation, in order to establish the distribution of these particles and their constituent apolipoproteins as a function of density. Lipoprotein subfractions were characterized by electrophorectic, chemical and morphological analyses, and their protein moieties were defined according to net charge at alkaline pH, molecular weight and isoelectric point. These analyses have permitted us to reevaluate the density limits of the major chicken lipoprotein classes and to determine their main characteristics, which are as follows: (1) very-low-density lipoproteins (VLDL), isolated at d less than 1.016 g/ml, were present at low concentrations (less than 0.1 mg/ml) in fasted birds; their mean diameter determined by gradient gel electrophoresis and by electron microscopy was 20.5 and 31.4 nm respectively; (2) as the the density increased from VLDL to intermediate density lipoproteins (IDL), d 1.016-l.020 g/ml) and low-density lipoproteins (LDL, d 1.020-1.046 g/ml), the lipoprotein particles contained progressively less triacylglycerol and more protein, and their Stokes diameter decreased to 20.0 nm; (3) apolipoprotein B-100 was the major apolipoprotein in lipoproteins of d less than 1.046 g/ml, with an Mr of 350000; small amounts of apolipoprotein B-100 were detectable in HDL subfractions of d less than 1.076 g/ml; urea-soluble apolipoproteins were present in this density range as minor components of Mr 38000-39000, 27000-28000 (corresponding to apolipoprotein A-1) and Mr 11000-12000; (4) high density lipoprotein (HDL, d 1.052-1.130 g/ml) was isolated as a single band, whose protein content increased progressively with increase in density; the chemical composition of HDL resembled that of human HDL2, with apolipoprotein A-1 (M 27000-28000) as the major protein component, and a protein of Mr 11000-12000 as a minor component; (5) heterogeneity was observed in the particle size and apolipoprotein distribution of HDL subfractions: two lipoprotein bands which additional apolipoproteins of Mr 13000 and 15000 were detected. These studies illustrate the inadequacy in the chicken of the density limits applied to fractionate the lipoprotein spectrum, and particularly the inappropriateness of the 1.063 g/ml density limit as the cutoff for LDL and HDL particle populations in the species.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Study of the atherogenic dyslipoproteinemia induced by dietary cholesterol in rhesus monekys (Macaca mulatta).

Hypercholesterolemia was induced in adult male rhesus monkeys with a high-fat diet containing an elevated cholesterol level (0.5%). Plasma lipoproteins were chromatographically separated into four size populations (regions) that were subdivided by density until fractions with single electrophoretic mobilities were obtained. The region III lipoproteins (LDL) contained 80% of plasma cholesterol and were present in the highest concentration of all fractions. Their molecular weight was increased over that of controls so that each particle averaged 1.8 times the number of cholesteryl ester molecules as did control LDL. Region II lipoproteins, a heterogeneous group, were present in next highest concentration. Most were cholesteryl ester-rich, beta-migrating lipoproteins that overlapped the VLDL and LDL density ranges; apoB was the predominant apoprotein. One region II subfraction had pre beta 2 migration and the density range. 1.050 less than d less than 1.10. Another subfraction, cholesteryl ester-rich VLDL including only about 1% of plasma cholesterol, had pre beta 1 migration and apoB and apoC as the predominant apoproteins with no apoprotein E. Region I lipoproteins were larger sized, slow beta-migrating cholesteryl ester-rich VLDL that included 5% of plasma cholesterol. ApoB and apoE were the predominant apoproteins. Region IV lipoproteins (HDL) contained 4% of the plasma cholesterol; their concentration was decreased to about 1/3 of the control level. Atherogenic features of the diet-induced dyslipoproteinemia included the increased plasma concentrations and cholesteryl ester contents of the region I, II, and III lipoproteins in addition to the decreased HDL concentration.     

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.     

Combined effects of desialylated and glycosylated low density lipoproteins on lipid content of human aortic intima cells in vitro]

We have studied a combined effect of glycosylated low density lipoproteins (LDL) on the cholesterol content of cells cultured from unaffected human aortic intima. Native LDL did not alter the intracellular cholesterol level while glycosylated LDL taken in the concentration of 50 and 100 mg/ml increased the cell cholesterol content by 30 and 70 percent, respectively. The effect of the same concentrations of glycosylated LDL treated with neuraminidase (desialylated-glycosylated LDL) was twice as powerful. Desialylated LDL in the concentration of 50 and 100 mg/ml raised the cholesterol level by 1.4- and 2.1-fold, respectively. Simultaneous incubation of cells with glycosylated (50 mg/ml) and desialylated (50 mg/ml) LDL brought about a 3.4-fold increase in intracellular cholesterol. The obtained data suggest that intensive development of atherosclerosis in diabetes mellitus may be partially explained by synergic effects of desialylated and glycosylated lipoproteins as well as LDL with both types of modification.     

Density distribution and physicochemical properties of plasma lipoproteins and apolipoproteins in the goose, Anser anser, a potential model of liver steatosis

The fractionation and physicochemical characterization of the complex molecular components composing the plasma lipoprotein spectrum in the goose, a potential model of liver steatosis, are described. Twenty lipoprotein subfractions (d less than 1.222 g/ml) were separated by isopycnic density gradient ultracentrifugation, and characterized according to their chemical composition, particle size and particle heterogeneity, electrophoretic mobility, and apolipoprotein content. Analytical ultracentrifugal analyses showed high density lipoproteins (HDL) to predominate (approximately 450 mg/dl plasma), the peak of its distribution occurring at d approximately 1.090 g/ml (F1.21 approximately 2.5). The HDL class displayed marked density heterogeneity, HDL1-like particles being detected up to a lower density limit of approximately 1.020 g/ml, particle size decreasing progressively from 17-19 nm at d 1.024-1.028 g/ml to 10.5-12 nm (d 1.055-1.065 g/ml), and then remaining constant (approximately 9 nm) at densities greater than 1.065 g/ml. HDL subfractions displayed multiple size species; five subspecies were present over the range d 1.103-1.183 g/ml with diameters of 10.5, 9.9, 9.0, 8.2, and 7.5 nm, four in the range d 1.090-1.103 g/ml (diameters 10.5, 9.9, 9.0, and 8.2 nm) and three over the range d 1.076-1.090 g/ml (diameters 10.5, 9.9, and 9.0 nm). ApoA-I (Mr 25,000-27,000) was the major apolipoprotein in all goose HDL subfractions, while the minor components (apparent Mr 100,000, 91,000, 64,000, 58,000, approximately 42,000, 18,000 and apoC-like proteins) showed marked quantitative and qualitative variation across this density range (i.e., 1.055-1.165 g/ml). The d 1.063 g/ml boundary for separation of goose low density lipoproteins (LDL) from HDL was inappropriate, since HDL-like particles were present in the density interval 1.024-1.063 g/ml, while particles enriched in apoB (Mr approximately 540,000) and resembling LDL in size (approximately 20.5 nm) were detected up to a density of approximately 1.076 g/ml. Goose LDL itself was a major component of the profile (90-172 mg/dl) with a single peak of high flotation rate (Sf approximately 10.5). The physicochemical properties and apolipoprotein content of intermediate density lipoproteins (IDL) and LDL varied but little over the range d 1.013-1.040 g/ml, presenting as two particle species (diameters 20.5 and 21 nm) of essentially constant chemical composition; LDL (d 1.019-1.040 g/ml) were separated from HDL1 by gel filtration chromatography and appeared to contain primarily apoB with lesser amounts of apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol

Human VLDL, LDL and HDL (very-low-, low- and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.