Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta).

A group of 14 adult male rhesus monkeys was maintained on a low cholesterol-high fat diet. Periodically, animals were fasted and blood samples were taken for characterization of the plasma lipoproteins. Complete separation of individual plasma lipoprotein classes was not achieved by traditional sequential ultracentrifugation techniques. Rather, initial separation of lipoprotein classes according to size was effected and density centrifugation was used subsequently for further separation. At least six lipoprotein fractions were identified, each of which was unique as defined by the properties of size, density (d), and electrophoretic mobility. These lipoprotein fractions were characterized by determination of chemical compositions and apoprotein patterns. The lipoproteins present in highest concentration in these monkeys were designated as region IV lipoproteins. This fraction had alpha-migration on agarose electrophoresis, 1.063 < d < 1.225, and the size, composition, and apoprotein pattern characteristic of HDL. No fewer than three fractions were identified with densities that overlapped the 1.019 < d < 1.063 range. Of these, the fraction designated as region III lipoproteins was present in highest concentration, had beta-migration by agarose electrophoresis, a predominant B apoprotein, and a chemical composition and size characteristic of LDL. Two larger subfractions, identified as region II lipoproteins, were separated from each other at a density of 1.050 g/ml. Agarose electrophoresis showed that the fraction with d < 1.050 had a migration intermediate between beta and pre-beta. The chemical composition and apoprotein pattern were consistent with the possibility that these lipoproteins were remnants of VLDL catabolism. The fraction with d > 1.050, had pre-beta mobility and a size and composition similar to the Lp(a) lipoprotein in plasma of human beings. At least two VLDL subfractions, identified as region I and IIa lipoproteins, were found although both were present in very low concentrations. Region I lipoproteins were larger and contained relatively more cholesteryl ester and more of the apoproteins that migrated with the mobility of apo-B and arg-rich apoprotein in SDS-polyacrylamide gel electrophoresis. Some of the region I lipoproteins were beta-migrating by agarose electrophoresis. These results suggested the possibility that a beta-migrating VLDL was present in these normal animals.     

In vitro incorporation of radiolabeled cholesteryl esters into high and low density lipoproteins

We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). Radiolabeled cholesteryl esters dissolved in absolute ethanol were mixed with LDL or HDL in the presence of lipoprotein-deficient serum (LPDS) as a source of core lipid transfer activity. The efficiency of incorporation was dependent on: a) the core lipid transfer activity and quantity of LPDS, b) the mass of added radiolabeled cholesteryl esters, c) the length of incubation, and d) the amount of acceptor lipoprotein cholesterol. The tracer incorporation was documented by repeat density gradient ultracentrifugation, agarose gel electrophoresis, and precipitation with heparin-MnCl2. The radiolabeling conditions did not affect the following properties of the lipoproteins: 1) chemical composition, 2) electrophoretic mobility on agarose gels, 3) hydrated density, 4) distribution of apoproteins on SDS gels, 5) plasma clearance rates, and 6) immunoprecipitability of HDL apoproteins A-I and A-II. Rat HDL containing radiolabeled cholesteryl esters incorporated in vitro had plasma disappearance rates identical to HDL radiolabeled in vivo.     

Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

Effect of dietary cholesterol on the lipoprotein profile and binding of radioiodinated lipoproteins to hepatic membranes in the cockerel (Gallus domesticus)

1. Cockerels fed a cholesterol-supplemented diet experienced a marked elevation of lipoprotein particles of density less than or equal to 1.006 g/ml (VLDL) and a diminution of lipoprotein particles of density 1.02-1.05 g/ml (LDL). 2. Unlike VLDL of some cholesterol-fed animals, cholesterol-fed cockerel VLDL did not display beta-mobility on agarose gel electrophoresis. 3. [125I]LDL and [125I]HDL binding to cockerel liver membranes was not affected by cholesterol feeding. 4. Different lipoprotein types appear to bind to a common site on cockerel liver membranes. 5. The results suggest that liver cells of cockerels may not possess LDL binding sites that are analogous to those of mammalian species.     

Comparison of two ultracentrifugation procedures for separation of nonhuman primate lipoproteins.

A comparison of nonhuman primate plasma lipoproteins isolated by swinging bucket rotor density gradient or fixed angle rotor differential ultracentrifugation is described. Whereas these two methods produced comparable results for the composition of low density (LDL) and high density (HDL) lipoproteins, the very low density lipoprotein (VLDL) fraction isolated with the swinging-bucket rotor contained relatively more cholesterol (free and esterified) and less phospholipid and protein than that fraction obtained with the fixed-angle rotor. Estimations of lipoprotein concentration by agarose gel electrophoresis and particle size by electron microscopy coupled with molar ratios of surface to core constituents indicate that the swinging bucket procedure resulted in a more complete harvest of VLDL particles, especially those in the larger size range.     

Ethanol induced alterations in low and high density lipoproteins

Male squirrel monkeys fed ethanol (ETOH) at variable doses were used to determine whether alcohol modifies levels of plasma low density lipoproteins (LDL) in addition to increasing high density lipoproteins (HDL). Because we earlier showed that high alcohol consumption enhances lipoprotein cholesterol synthesis, experiments were also performed to further assess whether ETOH alters lipoprotein clearance and plasma transfer processes in vivo. Monkeys were divided into three groups: Controls fed isocaloric liquid diet; and Low and High ETOH animals fed liquid diet with vodka substituted isocalorically for carbohydrate at 12 and 24 of the calories, respectively. High ETOH primates had significantly more LDL lipid and protein while serum glutamate oxaloacetate transaminase was similar for the three groups. Although removal of 3H LDL cholesteryl ester (CE) from the plasma compartment was not affected by dietary ETOH, transfer of LDL CE to HDL was impaired in the High ETOH group suggesting a mechanism for the enlarged circulating pool of LDL. Transfer of 14C HDL CE to lower density lipoproteins was similar for the three groups. However, ETOH at both doses delayed clearance of radiolabeled HDL CE from circulation. Thus besides enhancing synthesis of lipoproteins, ETOH at a moderately high dose (24% of calories) influences lipoprotein levels in primates by modifying lipid transfer processes (LDL) as well as by altering clearance (HDL) without adversely affecting liver function.     

Oral nicotine induces an atherogenic lipoprotein profile

Male squirrel monkeys were used to evaluate the effect of chronic oral nicotine intake on lipoprotein composition and metabolism. Eighteen yearling monkeys were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine primates given liquid diet supplemented with nicotine at 6 mg/kg body wt/day. Animals were weighed biweekly, plasma lipid, glucose, and lipoprotein parameters were measured monthly, and detailed lipoprotein composition, along with postheparin plasma lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activity, was assessed after 24 months of treatment. Although nicotine had no effect on plasma triglyceride or high density lipoproteins (HDL), the alkaloid caused a significant increase in plasma glucose, cholesterol, and low density lipoprotein (LDL) cholesterol plus protein while simultaneously reducing the HDL cholesterol/plasma cholesterol ratio and animal body weight. Levels of LDL precursors, very low density (VLDL) and intermediate density (IDL) lipoproteins, were also lower in nicotine-treated primates while total postheparin lipase (LPL + HTGL) activity was significantly elevated. Our data indicate that long-term consumption of oral nicotine induces an atherogenic lipoprotein profile (increases LDL, decreases HDL/total cholesterol ratio) by enhancing lipolytic conversion of VLDL to LDL. These results have important health implications for humans who use smokeless tobacco products or chew nicotine gum for prolonged periods.     

Non-denaturing polyacrylamide gradient gel electrophoresis for the diagnosis of dysbetalipoproteinemia

Dysbetalipoproteinemia, an uncommon but highly atherogenic mixed hyperlipidemia due to the accumulation of remnants of triglyceride-rich lipoproteins, is characterized by cholesterol-enriched VLDL that migrates in the beta-position on agarose gels. The demonstration of a broad beta-band on agarose gel electrophoresis of plasma is an insensitive method and ultracentrifugation is an impractical method of diagnosing this condition. Non-denaturing polyacrylamide gradient gel electrophoresis (PGGE) was investigated as a screening method for the diagnosis of dysbetalipoproteinemia. A minigel procedure separating the Sudan Black prestained apolipoprotein B (apoB)-containing lipoproteins on a 2-8% polyacrylamide gel at 4 degrees C overnight was analyzed for ultracentrifugally and genetically proven dysbetalipoproteinemic subjects as well as matched controls for mixed hyperlipidemia. Visual inspection revealed that the presence of only small VLDL- and IDL-like particles in untreated patients was highly sensitive (72%) and specific (95%) for dysbetalipoproteinemia. Videodensitometric analysis of area under the curve for large and small VLDL, as well as IDL and LDL, permitted even better discrimination in subjects whose profiles included some staining in the LDL-like region. A ratio of area under the curve of more than 0.5 for IDL-LDL allowed for a specificity of 100% and a sensitivity of 89% for the diagnosis of dysbetalipoproteinemia. This modified PGGE system may be useful in screening for dysbetalipoproteinemia.     

High density lipoprotein accumulation in perfusates of isolated livers of African green monkeys. Effects of saturated versus polyunsaturated dietary fat

To determine whether altered hepatic secretion of HDL is part of the mechanism by which polyunsaturated fat lowers plasma HDL concentration, we have studied HDL secretion in the isolated perfused livers of African green monkeys fed an atherogenic diet containing either safflower oil as the polyunsaturated fat or butter as the saturated fat. During recirculating perfusion with a lipoprotein-free medium, livers from safflower oil-fed animals produced 21% less HDL mass on the average than those from butter-fed animals. Newly secreted hepatic HDL were characterized after their isolation and subfractionation by a combination of agarose column chromatography and density gradient ultracentrifugation. In both diet groups the HDL were heterogeneous in size, morphology, and composition and consisted of discoidal particles ranging in diameter from greater than 200 A to as little as 50 A. Large, discoidal particles that were rich in apoE and apoA-I were separated from small particles that were poor in apoE but rich in apoA-I. All hepatic HDL subfractions contained only small amounts of cholesteryl ester and triglyceride. The hepatic particles resembled in composition and structure the large variety of HDL particles found in the plasma of patients with the familial deficiency of lecithin:cholesterol acyltransferase. Accordingly, perfusate LCAT activity was measured and found to be 2% or less than that in monkey plasma. We conclude that the perfused monkey liver produces a variety of nascent HDL that are relatively unmodified by the post-secretory metabolic events which normally occur in blood plasma in vivo, and that livers of polyunsaturated fat-fed monkeys secrete fewer plasma HDL precursor particles than do those of saturated fat-fed monkeys.     

Oral nicotine impairs clearance of plasma low density lipoproteins

The effect of chronic oral nicotine intake on plasma low density lipoprotein (LDL) clearance, lipid transfer protein, and lecithin:cholesterol acyltransferase (LCAT) was examined in male atherosclerosis susceptible squirrel monkeys. Eighteen yearling primates were divided into two groups: 1) Controls fed isocaloric liquid diet; and 2) Nicotine monkeys given liquid diet supplemented with nicotine at 6 mg/kg body wt/day for a two-year period. Averaged over 24 months of treatment, animals in the Nicotine group had significantly higher levels of plasma and LDL cholesterol compared to Controls while plasma LCAT activity was similar for both groups. Following simultaneous injection of 3H LDL and 14C high density lipoprotein (HDL) cholesteryl ester (CE), removal of the latter was not altered by oral nicotine while plasma clearance of 3H LDL was dramatically delayed in Nicotine monkeys. Transfer of 14C HDL CE to very low density lipoprotein (VLDL)-LDL particles was greatly accelerated in the Nicotine group vs Controls while the reciprocal movement of 3H LDL CE to HDL was only higher in experimental animals at two time points following injection of the isotopes. Results from this study provide evidence that one major detrimental effect of long-term oral nicotine use is an increase in the circulating pool of atherogenic LDL which is due to: 1) accelerated transfer of lipid from HDL; and 2) impaired clearance of LDL from the plasma compartment. Diminished removal of LDL is of particular importance because an extended residence time of these particles in circulation would increase the likelihood of their deposition in the arterial wall.     

The apo E/apo CIII molar ratio affects removal of cholesterol ester from modified human lipoproteins injected into cebus monkeys

The removal of postprandial (PP) and postabsorptive (PA) human LDL and HDL cholesterol was examined in cebus monkeys (Cebus albifrons) following in vitro labelling of these lipoproteins by 3H-cholesterol in the presence or absence of DTNB. The removal of LDL cholesteryl ester was 3.5 and 2 times greater than that of HDL in male and female monkeys, respectively. Incubation with DTNB reduced cholesteryl ester removal by 45 and 52% for LDL and HDL, respectively. Cholesteryl ester from PA lipoproteins was removed 80% faster than that PP particles only when plasma was incubated without DTNB. Cholesterol removal from these lipoproteins was positively (r = 0.941) and significantly (P less than 0.001) correlated with the molar apo E/apo CIII ratio. The data suggest that density of lipoproteins was less important than their apoprotein composition in dictating their removal from circulation.     

Heterogeneity of serum lipoproteins during the fetal and neonatal development of the pig

Serum lipoproteins from fetal, neonatal and adult pigs were characterized with the use of lipid analysis, polyacrylamide gradient gel electrophoresis, two-dimensional immunoelectrophoresis and zonal ultracentrifugation. Almost all serum cholesterol was found in LDL during the early stages of fetal development, while low but increasing levels appeared in the fetal pig HDL by the end of the gestation period. In the fetal pig, most of the serum triglycerides could be found in the HDL fraction. After the start of suckling, the levels of serum triglycerides and cholesterol increased. Most of these exogenous lipids were found in the chylomicrons + VLDL + LDL fraction of the newborn pig serum. The molecular weights of the native serum lipoproteins were calculated as being 2.0-2.4 X 10(5) daltons for newborn pig HDL and 1.4-1.7 X 10(6) daltons for newborn pig LDL. Minor changes in the molecular weight distributions were detected within these ranges for both HDL and LDL during fetal and neonatal development of the pig. Zonal ultracentrifugation of neonatal pig serum partly separated the LDL into three subfractions, whereas neonatal HDL appeared as one broad fraction.     

Lipopolysaccharide and tumor necrosis factor cause a fall in plasma concentration of lecithin: cholesterol acyltransferase in cynomolgus monkeys

The effects of intravenous injection of lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF) were investigated in cynomolgus monkeys (Macaca fascicularis). Injection of 20 micrograms/kg of LPS from E. coli (serotype 055:B5) into cynomolgus monkeys fed a monkey chow diet caused a twofold increase in plasma triglyceride and a 25% reduction in plasma cholesterol 48 h after injection. Similar results were found with injection of recombinant human TNF at a dose of 20 micrograms/kg into chow-fed animals. However, injection of the same dose of LPS or TNF into animals fed an atherogenic diet containing saturated fat and cholesterol resulted in a 2.4- to 5-fold increase in plasma triglyceride concentrations and no significant change in plasma cholesterol levels. The fall in plasma cholesterol levels observed in chow-fed animals was associated with a 57% decrease in the cholesteryl ester (CE) content in low density lipoprotein (LDL) and 35% decrease in CE in high density lipoprotein (HDL) in LPS-injected animals, and a decrease of 33% in CE concentration of LDL and 41% in CE of HDL in animals injected with TNF. In animals fed the atherogenic diet containing saturated fat and cholesterol, the injection of both LPS and TNF also resulted in a significant decrease in the CE content of LDL and HDL. However, the plasma total cholesterol levels did not change in the animals fed saturated fat and cholesterol because the decrease in CE content of LDL and HDL was offset by an increase in very low density lipoprotein (VLDL)-CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Density gradient characterization of the high density lipoproteins in cholesterol-fed hyper- and hyporesponding patas monkeys (Erythrocebus patas)

Diet-induced changes in high density lipoprotein (HDL) density and size were studied in patas monkeys. When the animals were switched from a moderate fat-low cholesterol diet to a high fat-high cholesterol (HFHC) diet, the plasma apoA-I levels increased initially in all of the animals. The apoA-I levels remained elevated in monkeys able to maintain their plasma cholesterol concentrations near basal levels (hyporesponders), but began to decrease in monkeys who became severely hypercholesterolemic (hyperresponders), reaching levels as low as 65-70% of their basal value by 24 weeks. The larger, lipid-rich HDL (HDL2) was shown by density gradient ultracentrifugation and gradient-PAGE (polyacrylamide gel electrophoresis) to be the HDL fraction responsible for these changes in apoA-I, completely accounting for the increase in apoA-I in hyporesponders and the decrease in apoA-I in hyperresponders. The HDL3 levels remained unchanged in hyporesponders but increased markedly in hyperresponders, partially compensating for the decrease of HDL2 in those animals. Gradient-PAGE showed the HDL3 to be heterogeneous, containing at least two populations of particles of the same density but differing significantly in size. The smaller of these HDL3 were most prominent in the HFHC-fed hyperresponders. These data show that nonhuman primate HDL is both physically and metabolically heterogeneous, and indicate that a high fat-high cholesterol diet-induced hypercholesterolemia severely depresses the HDL2 levels.     

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.     

Lipoprotein composition of human suction-blister interstitial fluid

Interstitial fluid (IF) was obtained in 27 apparently healthy subjects (12 males, 15 females) by applying mild suction (200-250 mm Hg) on the skin either on the midvolar forearm or on the paraumbilical region of the abdomen. The IF concentrations of lipids and apolipoproteins (apo) were studied and compared with those of serum (S). The mean ratio between interstitial fluid and serum (IF/S ratio) varied from 0.14 for forearm apoE to 0.29 for apoA-II on the abdomen. This ratio was consistently lower for apoE, C-II, C-III, and B than for apoA-I and A-II, and significantly lower on the arm than on the abdomen for all apolipoproteins studied. The IF/S ratios showed marked variations among individuals. However, interstitial fluid apolipoprotein concentrations at different blister sites were highly correlated within each individual. Studies with agarose gel electrophoresis and density gradient ultracentrifugation revealed that large triglyceride-rich particles were virtually lacking in interstitial fluid and that the relation between the low density lipoproteins (LDL) and high density lipoproteins (HDL) was shifted towards a greater proportion of HDL. The lipoprotein distribution in the HDL range of interstitial fluid differed from that of serum showing one maximum at a density of about 1.070 g/ml (serum HDL2 about 1.090 g/ml) and one at a density of 1.130-1.140 g/ml (serum HDL3, 1.110-1.120 g/ml). The former subfraction contained most of the lipoprotein-bound apoE while the latter contained the major part of apoA-I and apoA-II. Studies of the lipoproteins of interstitial fluid may add to our understanding of the development of atherosclerosis and xanthomatosis and may also provide valuable information on the permeability of the capillary membrane in normo- and pathophysiological states.     

Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

Polyclonal antibodies against iduronate 2-sulphate sulphatase from human urine

Low-density lipoproteins (density = 1.019-1.063 g/ml) were isolated in 10 subjects with type V hyperlipoproteinemia by ultracentrifugation in a zonal rotor under rate flotation conditions. Plasma LDL concentrations in these patients were extremely reduced, as well as being heterogeneous, and two different subclasses consisting of LDL2 (density = 1.019-1.045 g/ml) and LDL3 (density = 1.045-1.063 g/ml) were observed. LDL2 and LDL3 have similar electrophoretic mobilities in beta position in agarose gel, and their diameters, calculated from gel filtration studies, were inversely proportional to their densities. LDL2 and LDL3 have a mean hydrated density of 1.034 and 1.054 g/ml, respectively. In comparison with normal LDL2, the LDL2 and LDL3 of hypertriglyceridemic subjects are particularly rich in triacylglycerols and poor in cholesteryl esters and free cholesterol, while they have an increasing amount of proteins. The protein moiety is composed almost exclusively of apolipoprotein B-100 in IDL, LDL2 and LDL3 ; in addition, IDL also contain apolipoprotein C peptides. This characterization of LDL heterogeneity in type V hyperlipoproteinemia should be considered in interpreting kinetic data in human normal and pathological lipid metabolism and in evaluating the atherogenic risk of hypertriglyceridemia.     

A species comparison of low density lipoprotein heterogeneity in nonhuman primates fed atherogenic diets

Six male cynomolgus monkeys and five male African green monkeys were fed dietary cholesterol to induce hypercholesterolemia. The two groups studied had equivalent total plasma cholesterol concentrations. Low density lipoproteins (LDL) were isolated from whole plasma by ultracentrifugation and separated from other lipoprotein classes by agarose column chromatography. LDL were further subfractionated by density gradient ultracentrifugation in a VTi-50 vertical rotor. The material within five density regions was pooled from each sample and molecular weight, electrophoretic mobility, apoprotein heterogeneity, and percentage composition were determined for each subfraction. In general, cynomolgus monkey LDL were larger and more polydisperse than African green monkey LDL, and the LDL subfractions of cynomolgus monkeys were generally of lower densities although molecular weights at any density were in the same range for both species. ApoB-100 was the major apoprotein in each subfraction. ApoE was frequently present in the less dense subfractions while apoA-I was often seen in the more dense subfractions. Cynomolgus monkey LDL appeared to contain more apoE than African green monkey LDL. Over the entire spectrum of LDL, the percentage composition of the particles at any given density was indistinguishable between the species. In general, the average cynomolgus monkey LDL was larger, more polydisperse, less dense, and appeared to contain more apoE than the average African green monkey LDL. One or all of these differences might help explain the increased susceptibility to diet-induced atherosclerosis seen in cynomolgus monkeys.