Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

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.     

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.     

The role of the female Syrian hamster protein on the interaction between serum lipoproteins and heparin

We have previously shown the effect of phosphorylcholine-binding proteins from rat (PCBP) and rabbit (CRP) on the precipitation of serum lipoproteins by heparin in presence of Ca2+. The present paper describes the effect of a phosphorylcholine-binding protein from the female Syrian hamster (FP) on the lipoprotein precipitation reaction. The precipitation of lipoproteins by heparin was lower in assays using female hamster serum in which FP is a prominent protein, compared with assays with male serum in which FP is present in very low concentration. Depletion of FP from female serum resulted in increased lipoprotein precipitation. The addition of purified FP to assays using human very low density lipoprotein (VLDL) inhibited the precipitation reaction. The precipitation of lipoproteins was also examined using serum from male hamsters treated with diethylstilbestrol and female hamsters treated with testosterone, treatments known to modulate the levels of FP. Results indicate an inverse relationship between serum FP levels from normal and hormone-treated hamsters and the precipitation of lipoproteins from their serum. The partially desialylated FP when added to precipitation assays using human VLDL resulted in reduced inhibition of VLDL precipitation.     

Preventive medicine and family medicine in the medical school.

The normal variations in the paper electrophoretic lipoprotein patterns in 240 healthy Canadian males and females, aged 10 to 59 years, have been described and compared with serum cholesterol and triglyceride levels.The incidence of abnormal chylomicra, beta and pre-beta lipoproteins was similar in both sexes and increased with age in both sexes.Chylomicron bands and/or pre-beta trails from the origin occurred in 4% of subjects, pre-beta bands in 27% and “abnormally” dense beta bands in 28%.Five per cent of subjects were considered to have definite hyperlipoproteinemia, another 19% had slight and 21% had questionable hyperlipoproteinemia. Fifty-five per cent were normal.     

A comparison of some immunological characteristics of very low density lipoproteins of normal and hypercholesterolemic rat sera.

The immunological characteristics of a very low density lipoproteins (VLDL) from normal and hypercholesterolemic rat sera were compared using polyspecific antisera to VLDL and high density lipoproteins (HDL) and monospecific antisera to apo-B, apo-C, apo-A-I, and apo-E. Ultracentrifugally isolated VLDL from normal serum were studied by immunodiffusion and found to contain both discrete and associated (with apo-B) apo-C and apo-E, probably in the form of lipid-containing lipoproteins. However, immunoelectrophoresis of whole serum revealed only an associated form of the liporpotein having pre-beta mobility (i.e., VLDL), suggesting that the presence of discrete lipoproteins in isolated VLDL, each containing a single apoprotein family, may represent ultracentrifugal artifacts. Ultracentrifugally isolated VLDL from diet-induced hypercholesterolemic rat serum contained only trace amounts of apo-C and large quantities of apo-E, both of which were totally associated with apo-B. VLDL isolated by ultracentrifugation from perfusate of normal and hypercholesterolemic livers contained only associated lipoprotein complexes made up of apo-B, apo-C, and apo-E in the former but only apo-B and apo-E in the latter. These data suggest that normal VLDL are secreted as lipoprotein complexes containing apo-B, apo-C, and apo-E, which may become destabilized in the circulation. However, VLDL from hypercholesterolemic serum shows a marked diminution in the quantity of apo-C as indicated by the relative incorporation of [3H]leucine in vivo and by polyacrylamide gel electrophoresis of apo-VLDL.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Apolipoprotein A-I-containing lipoproteins with pre-beta electrophoretic mobility

Among the apoA-I-containing lipoproteins isolated by selected-affinity immunosorption from human serum and plasma, we have identified a subpopulation which, unlike the bulk of high density lipoproteins, has pre-beta electrophoretic mobility. This pre-beta subpopulation can be observed directly in fresh plasma by immunoelectrophoresis. It contains phospholipid and free and esterified cholesterol, but protein constitutes 90% of its mass. Apolipoprotein A-I is the predominant apolipoprotein in this subpopulation; apolipoprotein A-II and the B lipoproteins are not detected. The protein moiety of this subpopulation exhibits markedly lower helicity than that of high density lipoproteins isolated by ultracentrifugation.     

Characterization of very low density lipoprotein from Watanabe heritable hyperlipidemic rabbits

We investigated the properties of very low density lipoprotein (VLDL) from two types of Watanabe heritable hyperlipidemic (WHHL) rabbits: one with a high incidence of coronary atherosclerosis (type 1), and the other with a low incidence (type 2). When incubated with mouse peritoneal macrophages, VLDL from type 1 WHHL rabbit (type 1-VLDL) stimulated cholesteryl ester synthesis 10.5-fold more than VLDL from the type 2 WHHL rabbit (type 2-VLDL) did. Moreover, a similar difference was seen in the stimulation of cholesteryl ester synthesis in peritoneal macrophages isolated from the WHHL rabbits. The mass ratios of cholesterol to protein in type 1- and type 2-VLDL were 5.69 and 2.05, respectively. Agarose gel electrophoresis of type 1-VLDL showed beta mobility, and that of type 2-VLDL showed pre-beta mobility. No difference was seen between the sizes of VLDL particles of the two types. The amount of apolipoprotein E in type 1-VLDL was greater than that in type 2-VLDL. In conclusion, the difference between type 1 and type 2 WHHL rabbits is at least partly due to the presence in type 1 animals of VLDL particles rich in cholesteryl esters and apolipoprotein E, particles which are very similar to beta-VLDL in conformation.     

A novel phosphorylcholine-binding protein from rat serum and its effect on heparin-lipoprotein complex formation in the presence of calcium

An adsorbent was synthesized by attaching 4-aminophenylphosphorylcholine to cyanogen bromide-activated Sepharose. A phosphorylcholine (P-choline)-binding protein from rat serum was adsorbed on this affinity column which was eluted by 4 mM P-choline. The protein separated into two bands of Mr = 47,000 and 24,000 on sodium dodecyl sulfate-polyacrylamide gradient gels and contained 18% carbohydrate. A serum protein factor, precipitable by 30-50% (NH4)2SO4, was previously shown to inhibit Ca2+-heparin-rat serum very low density lipoprotein (VLDL) precipitation reaction, whereas P-choline counteracted the action of this protein (Mookerjea, S. (1978) Can. J. Biochem. 56, 746-752). It is now demonstrated that purified P-choline-binding protein prevents Ca2+-heparin-chylomicron or VLDL complex formation and P-choline reverses the effect of this protein. Antibody to P-choline-binding protein raised in rabbits produced a single precipitin line against the pure antigen. The antiserum, however, did not react against rat serum chylomicron, VLDL, low density lipoproteins, or high density lipoprotein. Human serum appears to lack P-choline-binding protein, since (a) the affinity column did not adsorb any such protein, (b) P-choline had no effect on the Ca2+-heparin-serum lipoprotein precipitation reaction, and (c) an immunodiffusion test against the antiserum was negative. However, when P-choline-binding protein was added to human serum, the lipoprotein precipitation was inhibited, and P-choline counteracted the effect of this protein. Preliminary experiments suggested a stoichiometric interaction between P-choline-binding protein and VLDL. Hydrophilic P-choline groups exposed on the surface of VLDL may possibly interact with the P-choline-binding protein and thereby affect the precipitation of lipoproteins by heparin and Ca2+.     

Characterization of human very low density lipoproteins containing two electrophoretic populations: double pre-beta lipoproteinemia and primary dysbetalipoproteinemia.

Two discrete populations of very low density lipoproteins, with fast and slow pre-beta electrophoretic mobility, were found in 50% of normolipemic and 30% of hyperlipemic individuals selected at random. The two populations were isolated by preparative electrophoresis from five hyperlipemic subjects. The particles comprising the slow component were smaller than those of the fast component and the slow component contained a larger proportion of cholesteryl esters, free cholesterol, B-apoprotein, and arginine-rich apoprotein and a smaller proportion of triglycerides and the two most anionic apoproteins (R-glutamic acid and R-alanine). The properties of the slow component thus closely resemble those of "remnant" very low density lipoproteins that accumulate in blood plasma of functionally hepatectomized rats. The chemical composition of the slow component was also similar to that of the very low density lipoproteins with beta mobility found in primary dysbetalipoproteinemia. However, the proportion of cholesteryl esters and argininerich apoprotein was much higher in the latter. The argininerich apoprotein from very low density lipoproteins of most normolipemic and hyperlipemic subjects separates into three or four major bands upon isoelectric focusing electrophoresis in polyacrylamide gels, with pI varying from 5.57 to 6.03. In very low density lipoproteins from individuals with primary dysbetalipoproteinemia, this protein uniquely contains little or none of the two most cationic bands. The number of bands was constant in all subjects studied. The pattern was the same in very low density lipoproteins with fast and slow pre-beta mobility as well as in the beta and pre-beta components in primary dysbetalipoproteinemia. These results suggest that many individuals have "remnant" very low density lipoproteins in their plasma. However, the beta-migrating "remnant" that accumulated in large amounts in individuals with primary dysbetalipoproteinemia contains much more arginine-rich protein and this protein is structurally abnormal.     

Hepatic perfusate very low density lipoproteins obtained from fat-fed nonhuman primates stimulate cholesterol esterification in macrophages

The livers of both baboons and rhesus monkeys fed a high fat, high cholesterol diet secreted very low density lipoproteins (VLDL) that were enriched in cholesteryl ester and apoe as compared to VLDL secreted by the livers of chow-fed animals. Stimulation of macrophage cholesterol esterification by the experimental VLDL was compared to that produced by the standard beta-VLDL obtained from the plasma of a rhesus monkey fed 25% coconut oil plus 2% cholesterol. This standard beta-VLDL stimulated 7- to 10-fold more esterification than did the bovine albumin control. Hepatic VLDL from fat-fed animals stimulated esterification in J774 macrophages 50 to 150% as well as did the standard beta-VLDL, even though hepatic VLDL did not display beta electrophoretic mobility on agarose gel electrophoresis. Plasma VLDL from lard-fed baboons did not exhibit beta electrophoretic mobility but did stimulate esterification in macrophages. Baboons were divided into high and low responders based on the change in plasma cholesterol levels in response to a high fat, high cholesterol diet. Both plasma and hepatic VLDL from high responders stimulated cholesterol esterification, whereas hepatic VLDL obtained from low responders or chow-fed baboons did not stimulate cholesterol esterification in macrophages. There was a strong positive correlation (r = 0.866) between the number of apoE molecules per VLDL particle in VLDL obtained from chow-fed, lard-fed, or coconut oil-fed primates and the rate of cholesterol esterification in macrophages. Our results show that hepatic perfusate VLDL obtained from fat- and cholesterol-fed primates have compositional and functional properties usually ascribed to circulating beta-VLDL, without displaying beta mobility, and indicate that the liver may be an important source of atherogenic lipoproteins.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of oral l-carnitine on lipoprotein composition in the Watanabe Heritable Hyperlipidemic Rabbit (Oryctolagus cuniculus)

1. We have recently reported the ability of orally administered l-carnitine to lower plasma triglyceride in the Watanabe Heritable Hyperlipidemic Rabbit (WHHL), an animal model of familial hyperlipoproteinemia. 2. In the present studies we examined the effect of l-carnitine administration upon individual lipoprotein subfractions in this animal model. 3. Carnitine feeding resulted in a reduction in very low density lipoproteins (VLDL) and high density lipoprotein (HDL). 4. Compositional analysis revealed a reduction in core triglyceride content with a concomitant increase in protein and phospholipid in VLDL and low density lipoproteins (LDL). 5. Conversely, electrophoretic mobility and apolipoprotein composition were unchanged with l-carnitine. 6. These results further demonstrate the ability of l-carnitine to modulate lipoprotein lipid composition in this animal model of familial hyperlipoproteinemia.     

Apoproteins of human serum high density lipoproteins. Isolation and characterization of the peptides of Sephadex fraction V from normal subjects and patients with abeta-lipoproteinemia.

1. Sephadex fraction V, obtained from human serum high density lipoprotein apoprotein (HDL apoprotein) of normal subjects and of patients with abetalipoproteinemia, was resolved by DEAE-cellulose ion exchange column chromatography into several fractions which were defined in terms of amino acid composition, NH2- and COOH-terminsls, sialic acid content, immunologic and electrophoretic properties, and in vitro activation of purified lipoprotein lipase from rat adipose tissue. 2. Fraction V of HDL apoprotein of both normal and abetalipoproteinemic subjects was found to contain polypeptides corresponding to apolipoproteins C-I, C-II, C-III-1, and C-III-2, which had been described previously in very low-density lipoproteins (VLDL). The content of apo C-III-1 in abetalipoproteinemia-HDL was very low, whereas the percentage, by weight, of apo C-I was about twice as high as that in the normal subjects studied. Furthermore, both normal and abetalipoproteinemia-HDL apoprotein contained a previously unreported peptide which had a molecular weight of about 7 000 and electrophoretic, chemical, and immunological properties distinct from those of the known C apolipoproteins. Of all of the peptides comprising fraction V, only apo C-II activated a purified preparation of rat adipose tissue lipoprotein lipase. This was the case for both normal and abetalipoproteinemic subjects.     

Concentration and composition of serum lipoproteins in the fetal rat]

1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.     

Effects of high cholesterol diets on rat plasma lipoproteins and lipoprotein-cell interactions

High fat, high cholesterol diets do not produce atherosclerotic lesions in some animal species such as the rat; however, when combined with experimentally induced hypothyroidism, such diets do produce lesions. While the diets or hypothyroidism each induce significant alterations in plasma lipoproteins, the combination produces marked hypercholesterolemia. If the atherosclerosis is related to the hyperlipidemia, the combination regimen could be provoking changes in the structure or compositions of lipoproteins which are not noted with either regimen alone. To test this hypothesis, Sprague-Dawley male rats (approximately 250 g) were treated as follows: Diet(a) = chow + 5% lard and 0.3% Na taurocholate; Diet(b) = Diet(a) + 2% cholesterol; Diet(c) = Diet(b) + 0.1% propylthiouracil (PTU). The major findings were as follows. 1) With Diet(b), slow floating very low density lipoprotein (VLDL) (pre-beta) enriched in cholesteryl esters accumulated in plasma and low density lipoprotein (LDL) disappeared from its usual flotation position. 2) With Diet(c), changes in plasma concentration were more marked but were also qualitatively different. More VLDL accumulated, and distribution of VLDL was shifted toward even slower floating cholesteryl ester-rich particles. VLDL had "broad beta" mobility. Also, a beta-migrating intermediate density lipoprotein (IDL) population appeared. 3) Lipoprotein (d less than 1.019 g/ml) and zonal subfractions of d less than 1.019 g/ml lipoproteins (isolated from rats on cholesterol Diet (b] stimulated [3H]oleate incorporation into cholesteryl esters of fibroblasts and macrophages, while the d less than 1.019 g/ml fractions of 5% fat (Diet(a]-fed rats did not. 4) The major finding of this study was that identically prepared d less than 1.019 g/ml fractions of Chol + PTU-treated rats (Diet(c] were approximately 2.5-fold more stimulatory than the lipoproteins of cholesterol-fed rats. The results could not be explained by differences in cholesterol contents of the cholesterol-rich lipoproteins, but significant differences in the apoprotein compositions of the fraction were found which could be important. The most active fractions had higher apoBL/apoBS and apoE/apoC ratios than less active fractions. Thus, the combination regimen of cholesterol and PTU produced changes in lipoprotein structure and composition which enhanced the abilities of the lipoproteins to interact with cells. The results suggest that analysis of lipoprotein-cell interactions in vitro may be predictive of the atherogenic potential of lipoproteins in vivo and that euthyroidism in rat protects against atherogenic hyperlipidemia.     

Isolation and characterization of lipoprotein of density less than 1.006 g/ml from rat hepatic lymph

The lipoprotein composition of rat hepatic lymph was studied using an animal model. The hepatic lymph duct of the adult male rat was cannulated and the hepatic lymph was collected. Hepatic lymph contains less than 1% of the total triglyceride output from the liver. Agarose gel electrophoresis of hepatic lymph showed the presence of two major lipoproteins bands, the alpha-migrating HDL and a band moving between plasma beta and pre-beta bands. Lipoprotein of density p less than 1.006 g/ml was then isolated by ultracentrifugation and it was found to correspond to the slow-moving pre-beta band. There was no difference in the mean diameter of hepatic lymph VLDL (64.4 nm) and that of plasma VLDL (64.6 nm). Compared with plasma VLDL, hepatic lymph VLDL has significantly more phospholipid (40% by weight), a higher cholesterol/cholesteryl ester ratio, and a marked reduction in triglyceride content (40% by weight). Although both plasma VLDL and hepatic lymph VLDL have apoE and apoB as the major apolipoproteins, there are other marked differences in apolipoprotein composition. Hepatic lymph VLDL has significantly less apoC and the apoB of hepatic lymph VLDL is predominantly the apoB240k (mol wt 240,000), with a small amount of the apoB330k (mol wt 335,000). On the other hand, plasma VLDL has an equal proportion of both apoB240k and apoB330k. This study presents for the first time the lipid and protein composition of rat hepatic lymph VLDL. Furthermore it has provided evidence that the hepatic lymph duct-cannulated rat can be used as an in vivo model for studying the secretion of nascent hepatic lymph VLDL.