Effect of fasting on the composition of plasma lipoproteins in cholesterol-fed diabetic rabbits

Cholesterol-fat feeding is associated with unusual alterations in the composition of plasma lipoproteins in alloxan-diabetic rabbits. In the present study plasma lipoprotein lipid and apoprotein composition was studied before and after 48 hr of fasting in cholesterol-fed diabetic and control rabbits in order to further characterize these alterations. Compared with control rabbits, the diabetic rabbits had similar plasma cholesterol levels, but 100-fold higher triglyceride levels prior to fasting. These plasma lipids were distributed mainly to large, Sf greater than 400 plasma lipoproteins in the diabetic rabbits, and to beta-VLDL in control rabbits. Sf greater than 400 lipoproteins, VLDL, IDL, LDL, and HDL from diabetic rabbits had triglyceride as the predominant lipoprotein core lipid. Sf greater than 400 lipoproteins and VLDL from diabetic rabbits had lesser amount of apoprotein E, and greater amounts of apoproteins A-I, A-IV, and B-48 as percent of total apoprotein mass in comparison with control rabbits. Fasting reduced plasma triglyceride levels by 55% in diabetic rabbits. Sf greater than 400 lipoprotein and VLDL triglyceride content decreased but remained a major core lipid. Fasting eliminated apoproteins A-I and A-IV from Sf greater than 400 lipoproteins and VLDL, but had no significant effect on apoB-48 content. Insulin treatment of the diabetic rabbits reduced plasma triglyceride by approximately 90% resulting in cholesteryl ester-rich particles reassembling beta-VLDL both in the Sf greater than 400 lipoprotein and VLDL fractions. These results indicate that the alterations in plasma lipoproteins in cholesterol-fed diabetic rabbits result from the presence in the d less than 1.006 g/ml plasma lipoprotein class of partially metabolized, intestinally derived particles.     

Metabolic heterogeneity in the formation of low density lipoprotein from very low density lipoprotein in the rat: evidence for the independent production of a low density lipoprotein subfraction.

The formation of low density lipoprotein (LDL) from very low density lipoprotein (VLDL) was studied after injecting 14C-radiomethylated or 125I-radioiodinated VLDL into rats. VLDL and LDL B apoprotein specific radioactivity time curves were obtained after tetramethylurea extraction of the lipoproteins. In all experiments, the specific activity of LDL B apoprotein did not intercept the VLDL curve at maximal heights, suggesting that not all LDL B apoprotein is derived from VLDL B apoprotein. Further subfractionation of LDL into the Sf 12-20, 5-12, and 0-5 ranges showed that most (65%) LDL B apoprotein was present in the Sf 0-5 fraction and that only a small proportion (6-15%) of this fraction was derived from VLDL. However, the curves obtained for the Sf 12-20 and 5-12 subfractions were consistent with a precursor-product relationship in which all of these fractions were derived entirely from VLDL catabolism. These results contrasted strikingly with similar data obtained for normal humans in which all LDL is derived from VLDL. In the rat, it appears that most of the B apoprotein in the Sf 0-5 range, which contains 65% of the total LDL B apoprotein, enters the plasma independently of VLDL secretion.     

Swine lipoproteins and atherosclerosis. Changes in the plasma lipoproteins and apoproteins induced by cholesterol feeding.

Cholesterol feeding in miniature swine resulted in a hypercholesterolemia with a distinctive hyperlipoproteinemia and the subsequent development of atherosclerosis. Alterations in the type and distribution of plasma lipoproteins induced by cholesterol feeding were as follows: (a) the occurrence of beta-migrating lipoproteins (B-VLDL) as well as very low density lipoproteins in the d less than 1.006 ultracentrifugal fraction; (b) an increased prominence of the intermediate lipoproteins (d = 1.006-1.02); (c) an increased prominence of low density lipoproteins; and (d) the occurrence of a distinctive lipoprotein with alpha mobility which was referred to as HDLc (cholesterol induced). Characterization of the various plasma lipoproteins included chemical composition, size by electron microscopy, and apoprotein content. The B-VLDL resembled the beta-migrating lipoproteins of human Type III hyperlipoproteinemia and contained a prominent protein equivalent to the arginine-rich apoprotein in addition to the B apoprotein, apo-A-I, and the fast-migrating apoproteins (apo-C). The HDLc were rich in cholesterol, ranged in size from 100 to 240 A in diameter, and contained the arginine-rich apoprotein and apo-A0I but lacked the B apoprotein. The arginine-rich apoproteins isolated from B-VLDL and HDLc by gel chromatography were similar in amino acid analyses, with glutamic acid as their amino-terminal residue. The occurrence of a spectrum of cholesterol-rich lipoproteins which contained the arginine-rich apoprotein with the occurrence of accelerated atherosclerosis suggested an interesting, although speculative, association.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

A study on the selective binding of apoprotein B- and E-containing human plasma lipoproteins to immobilized rat serum phosphorylcholine-binding protein

Rat serum phosphorylcholine-binding protein (PCBP), a member of the pentraxin family of proteins, was previously shown to bind multilamellar liposomes prepared with egg phosphatidylcholine and lysophosphatidylcholine. The results suggested that the phosphorylcholine groups on the surface of liposomes play an important role in the binding process (Nagpurkar, A., Saxena, U., and Mookerjea, S. (1983) J. Biol Chem. 258, 10518-10523). A study on the binding of human plasma lipoproteins to PCBP immobilized on Sepharose has now been initiated. Very low density lipoproteins were partially bound to a Sepharose-PCBP column, and the bound fraction contained higher concentrations of apoprotein B and E. All the low density lipoproteins applied were bound to the column. In the case of high density lipoproteins, only a small fraction was retained on the column (based on protein analysis), and that bound fraction contained all the apoprotein E and Lp(a) lipoprotein. The binding of very low, low, and high density lipoproteins to Sepharose-PCBP was Ca2+-dependent, and the bound lipoproteins were quantitatively eluted by a phosphorylcholine gradient. Apoprotein B and E were also bound when whole human plasma was applied to Sepharose-PCBP. The effect of selective modification of lysine residues by acetoacetylation and of arginine residues by cyclohexanedione on the binding of low density lipoproteins to Sepharose-PCBP was examined. Modification of arginyl residues resulted in marked reduction of binding, whereas modification of lysine had no effect. Removal of sialic acid from PCBP also had no effect on the binding of low density lipoproteins to immobilized-desialylated PCBP column. The preferential binding of apoprotein B- and E-containing lipoproteins to Sepharose-PCBP indicates a possible physiological role of PCBP and other similar circulating phosphorylcholine-binding proteins of the pentraxin family in lipoprotein metabolism.     

Inhibition of cholesterol synthesis reduces low-density-lipoprotein apoprotein B production without decreasing very-low-density-lipoprotein apoprotein B synthesis in rabbits.

The kinetics of the apoprotein B (apo B) of very-low-density (VLDL; d less than 1.006) and low-density (LDL; d 1.019-1.063) lipoproteins were studied in six rabbits by using radioiodinated homologous lipoproteins, before and during oral administration of mevinolin (5 mg/kg per day), a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (EC 1.1.1.34), to explore the mechanism by which the drug reduces LDL synthesis. Before treatment LDL-apo B production greatly exceeded VLDL-apo B production in all animals, indicating that a large proportion of plasma LDL was derived from a VLDL-independent pathway. Five animals responded to mevinolin with a fall in plasma cholesterol (mean change - 53%; P less than 0.01). This was associated with a 66% decrease in LDL-apo B synthesis (P less than 0.05). In contrast, VLDL-apo B synthesis was unaffected by mevinolin. Furthermore, in all but one animal the decrement in LDL-apo B synthesis was greater than the rate of VLDL-apo B synthesis before treatment, demonstrating that mevinolin had reduced the VLDL-independent production of LDL.     

Large lipoproteins are excluded from the arterial wall in diabetic cholesterol-fed rabbits

In diabetic hypercholesterolemic rabbits at plasma triglyceride concentrations of approximately 5000 mg/dl, 55% of plasma cholesterol (1400 mg/dl) was in lipoproteins with diameters larger than 75 nm (Sf greater than 400), 40% in smaller very low density and intermediate density lipoproteins, 4% in low density lipoproteins, and 1% in high density lipoproteins. Specific intimal clearance (nl/h.mg aortic cholesterol) of the giant Sf greater than 400 lipoproteins was about 4% of that of the low density lipoproteins. The data suggest that even very low density lipoproteins with diameters smaller than 75 nm were practically excluded from entering the arterial wall. Specific intimal clearance of low density lipoproteins in hypertriglyceridemic, diabetic cholesterol-fed rabbits was similar to that in normal cholesterol-fed rabbits, but low density lipoprotein concentrations in diabetic rabbits were low. Thus, at plasma triglyceride concentrations of approximately 5000 mg/dl, only 5% of plasma cholesterol may be readily available for infiltration of arteries. These results add further support to the hypothesis that hypertriglyceridemic, diabetic cholesterol-fed rabbits are protected against atherogenesis because the major part of plasma cholesterol is carried in large lipoproteins to which the artery is not very permeable.     

Changes in plasma very low density and low density lipoprotein content, composition, and size after a fatty meal in normo- and hypertriglyceridemic man.

Four subfractions of plasma VLDL characterized by decreasing Sf value and LDL were isolated by density gradient preparative ultracentrifugation from normotriglyceridemic (NTG) and hypertriglyceridemic (HTG) (type IV) subjects in the fasting state and after a fatty meal. Chemical analysis and computation of numbers of particles in each fraction showed that the hyperlipidemia of type IV subjects was accounted for by an increase in total numbers of VLDL and a shift in the distribution of VLDL towards particles of larger diameter. Postprandial hyperlipidemia was due to the presence of chylomicron remnants rather than intact chylomicrons, and was accounted for by an increase in particle diameter of the largest VLDL subfraction rather than by an increase in particle numbers. Postprandial hyperlipedemia was accompanied by a shift in the distribution of VLDL towards particles of larger diameter in both NTG and HTG subjects, probably because of competition for the triglyceride-depletion process between chylomicrons and hepatic VLDL. Most chylomicron remnants were removed from the circulation without degradation to smaller VLDL or to LDL, but some remnants were sufficienty small to contribute to smaller VLDL subfractions. The LDL of type IV subjects contained more apoprotein B than those from NTG subjects, and this difference was associated with increases in diameter, molecular weight, density, and the ratio of protein: phospholipid in LDL from type IV subjects. Defective degradation of large VLDL to small VLDL, and of VLDL to LDL may be related to this alteration in apoprotein B content of the lipoproteins in type IV subjects.     

Characterization of plasma lipoproteins of grain- and cholesterol-fed White Carneau and Show Racer pigeons

Plasma cholesterol concentrations from White Carneau (WC) and Show Racer (SR) pigeons consuming a cholesterol-free grain diet averaged about 300 mg/dl, approximately 200 mg/dl as high density lipoproteins (HDL) and the remainder as low density lipoproteins (LDL). Consumption of a cholesterol-containing diet increased plasma cholesterol concentrations in both breeds to greater than 2000 mg/dl. Approximately one-half of this increase was as LDL with the remainder as beta-migrating very low density lipoproteins (beta-VLDL). There was little change in HDL concentration. LDL from cholesterol-fed animals had a greater net negative charge than control LDL, and was larger (Mr = 10 X 10(6) vs 3.2 X 10(60)) due to an increase in the number of cholesteryl ester molecules per particle. The principal apoprotein of LDL was apoB-100 with smaller amounts of apoA-I and several minor unidentified apoproteins. beta-VLDL was cholesteryl ester-rich, could be separated into two size populations by gel chromatography, and contained apoB-100 as its principal apoprotein. Apoprotein E was not detected in any of the plasma lipoproteins. HDL from control and cholesterol-fed animals was composed of a single class of particles with virtually identical composition resembling HDL2. The major apoprotein of HDL was apoA-I. There were no consistent quantitative or qualitative differences in the lipoproteins of the two breeds of pigeons that could help to explain the susceptibility to atherosclerosis of the WC or the resistance of the SR.     

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.     

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.     

The binding of very low density and low density lipoproteins to the plasma membrane of the hen's oocyte : A morphological study

The binding of lipoproteins to the oocyte plasma membrane of the domestic fowl (Gallus domesticus) was examined by electron microscopy in preparations of the ovarian follicle in the main phase of yolk formation. Numerous particles, 26 nm in diameter, were present on the untreated membrane. They were dissociated from the membrane by incubation at 4 °C in buffer at pH 6.2 and with heparin at pH 7.4. Added calcium was not required for binding, though the number of bound particles was reduced by treatment with EDTA. Very low density (VLD) lipoproteins from laying hen's plasma were found to bind to the denuded membrane and to correspond in size to the native particles. The results suggest that the binding characteristics are similar in quality to those determined for the binding of low density (LD) lipoproteins to mammalian cells. The oocytes, however, bound 100-fold more particles per unit length of membrane. VLD and LD lipoproteins from immature hens also adhered to the denuded membrane, although their apoprotein composition was very different from that of laying hen VLD lipoproteins. LD lipoproteins from immature hens and VLD lipoproteins from laying hens both contained apo-B, which formed about 80 and 35%, respectively, of the total apolipoproteins. Apo-VLDL-II is the other major apoprotein identified in laying-hen VLD lipoproteins. Apo-VLDL-II was not positively identified as a component of immature-hen LD lipoproteins and could only have been present as a minor component. Despite their great difference in apo-VLDL-II content, immature-hen LD lipoproteins and laying-hen VLD lipoproteins showed similar dissociation constants for binding to the oocyte plasma membrane. This evidence strongly suggests that the cell surface receptors recognize the B apoprotein of avian VLD lipoproteins.     

Enhanced activation of bound plasminogen on Staphylococcus aureus by staphylokinase

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

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.     

Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.

Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL.     

Abnormal clearance of postprandial Sf 100-400 plasma lipoproteins in insulin-dependent diabetes mellitus

Studies were carried out in three normolipidemic non-obese men with insulin-dependent diabetes mellitus (IDDM) and three normal men, to assess whether the clearance of postprandial Sf 100-400 lipoproteins is decreased in IDDM. Sf greater than 100 lipoproteins isolated from plasma 4.5 h after fat ingestion were labeled with 125I and injected into the same subject intravenously. ApoB radioactivity was measured over time in Sf greater than 400, Sf 100-400, and Sf 20-100 lipoproteins isolated from plasma and analyzed using a kinetic model that included both fast and slow delipidation cascades, where lipolysis and uptake of particles by the liver and other tissues were represented. Fractional catabolic rates of Sf 100-400 lipoproteins (min-1) were decreased in diabetic versus control subjects: fast = 0.170 +/- 0.126 versus 0.680 +/- 0.242 (mean +/- SD) (P less than 0.05, two-tailed) and slow = 0.011 +/- 0.006 versus 0.031 +/- 0.015 (P less than 0.05, one-tailed). Kinetic analysis showed that the data were consistent with decreased uptake by the tissues for the fast cascade (diabetic, 0.084 +/- 0.082, vs. control, 0.617 +/- 0.328, P less than 0.05, one-tailed). A similar trend was observed for the slow cascade. There were no significant differences between the two groups in the intraplasma lipolysis rates of Sf 100-400 particles. Analysis of the composition of the injected particles showed that they were total cholesterol (TC)- versus triglyceride (TG)-enriched (P less than 0.001, log-ratio analysis of composition) in IDDM subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a human plasma cholesteryl ester transfer protein

The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and promotes transfer of cholesteryl esters between the lipoproteins. CETP has been purified 55,000-fold, with a 27% recovery of activity, from the d greater than 1.21 g/ml fraction of human plasma. In the final purification step, partially purified CETP is incubated with a synthetic lipid emulsion consisting of phosphatidylcholine, triglyceride, and fatty acid, and the bound activity, which elutes in the void volume, is separated from nonbound proteins by gel filtration on Sepharose 4B. Sodium dodecyl sulfate-gel analysis of fractions containing bound activity shows the presence of a single protein with an apparent Mr of 74,000. Inclusion of fatty acid in this emulsion was required to prevent the binding of a contaminant protein. However, incubation of CEPT with fatty acid emulsions containing lipid peroxides resulted in substantial inactivation and covalent degradation of the 74-kDa protein. This could be prevented by the inclusion of antioxidants during preparation of the emulsion. Solvent extraction of emulsion-bound CEPT gave a delipidated, active preparation. Purified IgG from a rabbit immunized with the 74-kDa protein completely removed activity from partially purified fractions. Amino acid analysis of the purified protein showed it to contain an unusually high content (45%) of nonpolar residues; the calculated hydrophobicity was greater than that of any other plasma apolipoprotein. These results show human CETP to be a unique plasma apolipoprotein with an apparent Mr of 74,000 which is hydrophobic, self-associating, and susceptible to covalent degradation by lipid peroxides.     

The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout.

1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history.