The composition and metabolism of high density lipoprotein subfractions

The composition and metabolism of high density lipoprotein (HDL) subfractions were investigated in seven nomal individuals. Mean HDL₂ (d, 1.063–1.125 g/ml) composition (by weight) was 43% protein, 28% phospholipid, 23% cholesterol, and 6% triglyceride, and mean HDL₃ (d, 1.125–1.21 g/ml) composition was 58% protein, 22% phospholipid, 14% cholesterol, and 5% triglyceride. The mean apoA-I; apoA-II weight ratio was 4.75 for HDL₂ and 3.65 for HDL₃.HDL₂ protein was proportionally slightly richer in C apolipoproteins and higher molecular weight constituents (including apoE) than HDL₃. Kinetic studies utilizing radiolabeled HDL_A (d, 1.09–1.21 g/ml), HDL₂, and HDL₃ demonstrated rapid exchange of apoA-I and apoA-II radioactivity among HDL subfractions, similar fractional rates of catabolism of apoA-I and apoA-II within HDL, and similar radioactivity decay within HDL subfractions. Mean plasma residence time was 5.74 days for radiolabeled HDL₂ and 5.70 days for radiolabeled HDL₃. Differences in HDL protein mass among individuals were largely due to alterations in catabolism, and in general both HDL₂ and HDL₃ were catabolized via a plasma and a nonplasma pathway. Data from simultaneous radiolabeled very low density lipoprotein and HDL studies in 2 individuals are consistent with the concept that apoC-II and apoC-III are catabolized at a different rate than are apoA-I and apoA-II within the HDL density range.     

Serum lipoprotein and apoprotein concentrations in 4-(4-chlorophenyl)-2-hydroxytetronic acid and clofibrate-treated cholesterol and cholic acid-fed rats

Influence of clofibrate and an aci-reductone, 4-(4-chlorophenyl)-2-hydroxytetronic acid (CHTA) on lipoproteins and apoproteins was studied in cholesterol- plus cholic acid-fed rats. CHTA (0.4 mmol/kg body wt, twice daily) significantly lowered serum total cholesterol and triglyceride concentrations at both 10 and 16 days, whereas clofibrate at the same dose did not alter serum cholesterol levels, but elevated serum triglyceride concentrations at 16 days. The abnormal cholesterol-rich very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL) produced by cholesterol plus cholic acid were significantly reduced in their cholesterol content by treatment with CHTA, a compound having an oxidation reduction potential. Conversely, clofibrate administration increased VLDL-cholesterol with concomitant decreases in IDL- and LDL-cholesterol concentrations. Administration of CHTA to cholesterol- plus cholic acid-fed rats significantly increased concentrations of VLDL and IDL, but had no effect on HDL protein. Both CHTA and clofibrate administration to cholesterol- plus cholic acid-fed rats significantly lowered IDL protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies of apoproteins revealed that clofibrate treatment significantly reduced apoC-III and C-II in VLDL, C-II in IDL, and apoA-IV and A-I in HDL. Rats treated with CHTA significantly raised apoC-II and C-III in HDL. Isoelectric focusing (IEF) of VLDL apoproteins showed a significant decrease in apoC-II, C-III-0 and apoC-III-3 in clofibrate-treated animals. Thus, the mechanism for antilipidemic action of the oxidation reduction compound, CHTA, which differs markedly from the prototype drug, clofibrate, is independent from major apoprotein modification.     

ApoA-I secretion by rabbit intestinal mucosa cell cultures

Lipid and apolipoprotein (apo) A-I concentrations in different density fractions of New Zealand White (NZW) and Watanabe (WHHL) rabbit plasma were studied. Aside from the low plasma apoA-I and high density lipoprotein (HDL) cholesterol levels in WHHL rabbits, the distribution of apoA-I was also different between the two rabbits. ApoA-I was concentrated in both the HDL₂ and HDL₃ fractions of NZW rabbits but was found primarily in the HDL₃ fraction of WHHL rabbits. ApoA-I secretion in these two rabbits was further studiedin vitro by using intestinal and hepatocyte cell cultures. ApoA-I secretion was highest from cultures of the duodenum and the proximal end of the jejunum; whereas, cell cultures of the distal end of the small intestine secreted very little apoA-I into the medium. Intestinal cell cultures from WHHL rabbits secreted less, but significant amounts of, apoA-I compared to that of NZW rabbits. ApoA-I was most concentrated in the density range of 1.12–1.21 (HDL₃) fraction in medium containing 10% fetal calf serum (FCS). Serum-free medium promoted apoA-I secretion by intestinal cell cultures that was mostly found in the d>1.21 (lipoprotein-deficient) fraction. Hepatocytes isolated from the same rabbits by collagenase perfusion secreted little apoA-I, and it was found only in the d>1.21 fraction. The addition of oleic acid into the culture medium with 10% FCS decreased the secretion of total apoA-I and HDL by intestinal cell cultures and increased the secretion of very low density lipoprotein (VLDL) and intermediate density lipoproteins (IDL). The results indicate that intestinal cells, not hepatocytes, are responsible for the secretion of apoA-I and HDL₃ in rabbits, and that the secretion may be regulated under different nutritional conditions.     

Changes in high density lipoprotein subfraction lipids during neutral lipid transfer in healthy subjects and in patients with insulin-dependent diabetes mellitus

While it is known that the transfer of cholesteryl ester (CE) from high density lipoprotein (HDL) to the apo B-containing lipoproteins is increased in patients with diabetes, the extent to which the various lipoprotein fractions engage in neutral lipid exchange and the magnitude to which triglyceride (TG) is translocated is not known. To examine in greater detail neutral lipid net mass transfer in diabetes, the HDL subfractions and the apo B-containing lipoproteins were separated, and the net mass transfer of CE and TG was compared to that of control subjects. In both groups, bidirectional transfer of CE from HDL₃ to very low density lipoprotein (VLDL) + low density lipoprotein (LDL) and of TG from VLDL+LDL to HDL₃, took place, but this process was significantly greater (P<.01) in insulin-dependent diabetes mellitus (IDDM). In contrast, CE and TG accumulated in HDL₂ to a similar degree in normal and IDDM subjects. In recombination experiments with each of the apo B-containing lipoproteins, IDDM VLDL had a greater capacity to facilitate the exchange of core lipids from both IDDM and control HDL₃: on the other hand, LDL from IDDM and control subjects both donated TG and CE to HDL₂ and affected little change in HDL₃. These findings indicate that all the major plasma fractions normally participate in the trafficking of CE and TG among the lipoproteins during neutral lipid transfer and show that the principal perturbation in cholesteryl ester transfer in IDDM involves altered interaction between VLDL and the HDL₃ subfraction.     

Rapid fractionation of human high density apolipoproteins by high performance liquid chromatography

A simple and rapid fractionation procedure (30 min) has been developed for the isolation of the major apoproteins from human serum high density lipoproteins by molecular sieving in a high performance liquid chromatographic column. Apo A-I, apoA-II and the C peptides are quantitatively resolved up to a protein load of 3 mg. The technique has also been successfully applied to the final purification of A apoproteins which has been isolated by conventional chromatographic procedures and as a sensitive analytical tool for assessing apoprotein purity.     

Interaction of plasma high density lipoprotein HDL2b (d 1.063–1.100 g/ml) with single-bilayer liposomes of dimyristoylphosphatidylcholine

Incubation of a major subfraction, HDL_2b (d 1.063–1.100 g/ml), of human plasma high density lipoproteins, HDL (d 1.063–1.21 g/ml), with single-bilayer liposomes of dimyristoylphosphatidylcholine (DMPC) resulted in uptake of DMPC by the HDL_2b and dissociation of lipid-free apolipoprotein A-I (apoA-I). In the presence of excess DMPC, the dissociated apoA-I was also incorporated with DMPC into discoidal complexes. Preliminary studies with model apoA-I-DMPC complexes indicated that they also can interact with native HDL_2b with the resultant transfer of their DMPC to HDL_2b and the concomitant release of their apoA-I. After interaction of HDL_2b with DMPC liposomes, the DMPC-enriched HDL_2b product showed a lower hydrated density and a larger particle size than the control HDL_2b. The molecular properties of the lipoprotein product suggest that stabilization of the apoA-I-depleted HDL_2b probably occurred via substitution of DMPC for the apoA-I at the HDL_2b surface rather than by fusion of the apoA-I-depleted HDL_2b. The above interactions of HDL_2b with single-bilayer liposomes and discoidal complexes indicate pathways of phospholipid transfer relevant to the possible role of HDL in the metabolism of lipoprotein surface components in vivo.     

Glycosphingolipids of human plasma lipoproteins

The content and structure of glycosphingolipids (GSL) in human plasma lipoproteins were studies. The quantitative distribution of the neutral GSL(Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer) and the principal ganglioside (AcNeu-Gal-Glc-Cer) within the different lipoprotein classes was similar to that of whole plasma. The total amounts (μmol glucose/100 ml plasma) of GSL in the plasma lipoproteins of three normal subjects were VLDL (very low density lipoproteins) (trace to 0.46), LDL (low density lipoproteins) (1.08–1.48), HDL₂ (high density lipoproteins₂) (0.62–0.85), and HDL₃ (high density lipoproteins₃) (trace to 0.28). In subjects with Lp(a) lipoproteins, HDL₂ rather than HDL₃ contained most of the GSL in HDL. When the data were corrected for differences in the plasma concentrations of the lipoproteins, the total amounts of GSL(nmol glucose/mg lipoprotein cholesterol) were VLDL(trace to 21.20), LDL(11.70–15.36), HDL₂(8.50–9.10), and HDL₃(3.12). No GSL were detected in lipoprotein deficient plasma. Mass spectrometry of the trimethylsilyl derivatives of the GSL in LDL showed major fragment ions characteristic of their individual structural components. The elevated plasma levels of the GSL(2–18 fold), in a homozygote for familial hypercholesterolemia, resided in LDL which contained an absolute increase (per mg lipoprotein cholesterol) of GSL. Most, if not all, of the plasma GSL are associated with plasma lipoproteins and may have an important role in their biological functions.     

Net lipid transfer between lipoproteins in fish-eye disease plasma supplemented with normal high density lipoproteins

Native fish-eye disease plasma, which is deficient of both high density lipoproteins (HDL) and lecithin-cholesterol acyltransferase activity (α-LCAT), processing the free cholesterol of these lipoproteins, has been supplemented with normal isolated HDL₂ or HDL₃ and incubated in vitro at 37 C. After incubation for 0,7.5 and 24 hr the very low density (VLDL) and low density (LDL) lipoproteins as well as HDL were isolated, and their contents of triglycerides, phospholipids and free, esterified and total cholesterol were quantified. The resulting net mass transfer of the different lipids revealed a functioning transfer of cholesteryl esters and all other analyzed lipids between the lipoproteins, although no de novo esterification of the HDL cholesterol by LCAT in this plasma occurred. In accordance with previous findings there was a functioning esterification process of the free cholesterol of the combined VLDL and LDL of fish-eye disease plasma. The present results make it reasonable to conclude that the lack of HDL cholesterol esterification in this disease is not a result of a deficiency of cholesteryl ester transfer or lipid transfer activities.     

The influence of plasma apolipoprotein A‐II concentrations on HDL subclass distribution

Background and aims: To investigate the impact of plasma apoA‐II concentrations on the alteration of HDL subclass distribution, and the cooperative effect of apoA‐I and apoA‐II on it. Methods and results: The apoA‐I contents of plasma HDL subclasses were quantified by two‐dimensional gel electrophoresis associated with immunodetection for 292 Chinese people. These subjects were divided according to the mean ± 1 SD of apoA‐II and apoA‐I levels as two cut‐points, respectively. Compared with the low‐apoA‐II group, the apoA‐I contents of HDL_3a (in the high group), HDL_3b, and HDL_2b increased strikingly, both in the middle‐ and high‐apoA‐II group. The apoA‐I contents of all HDL subclasses increased progressively when the apoA‐I and apoA‐II levels simultaneously or the apoA‐I/apoA‐II ratio increased, and in comparison to the low‐apoA‐I–A‐II levels group, the apoA‐I contents of HDL_2b (115%) increased more significantly than those of preβ₁‐HDL (39%) in the high‐apoA‐I–A‐II levels group. Multiple analyses also indicated that the three HDL subclasses, HDL_3a, HDL_3b and HDL_2b, were independently predicted by apoA‐II. Conclusion: Excess apoA‐II can cause the accumulation of both large‐sized HDL_2b and small‐sized HDL₃, which implies that apoA‐II plays a double role in the HDL maturation metabolism. Meanwhile, the degree of HDL_2b increased significantly relative to that of preβ₁‐HDL when apoA‐I and apoA‐II levels were elevated simultaneously, suggesting that the maturation and metabolism of HDL might be promoted and reverse cholesterol transport might be enhanced.     

Effect of a salmon diet on the distribution of plasma lipoproteins and apolipoproteins in normolipidemic adult men

The effects of n−3 fatty acids on plasma lipids, lipoproteins and apoproteins have usually been studied in humans after feeding of purified fish oil. This study describes the effect of a natural diet, containing salmon as the source of n−3 fatty acids, on these parameters as compared to a diet very low in n−3 fatty acids. The subjects were nine normolipidemic, healthy males who were confined to a nutrition suite for 100 days. During the first 20 days of the study the participants were given a stabilization diet consisting of 55% carbohydrates, 15% protein, and 30% fat. The n−3 content of this diet was less than 1%, and it contained no 20- or 22-carbon n−3 fatty acids. After the stabilization period the men were split into two groups, one group continued on the stabilization diet while the other received the salmon diet that contained approximately 2.1 energy percent (En%) of calories from 20- and 22-carbon n−3 fatty acids. Both diets contained equal amounts of n−6 fatty acids. This regime continued for 40 days, then the two groups switched diets for the remainder of the study. Plasma triglycerides were lowered significantly (p<0.01) and high density lipoprotein cholesterol (HDL-C) was significantly elevated (p<0.01) after the men consumed the salmon diet for 40 days. The very low density lipoproteins (VLDL) were lowered, but the trend did not reach statistical significance during the intervention period. The total plasma cholesterol, total low density lipoprotein (LDL) and the total high density lipoprotein (HDL) levels were not influenced by the salmon diet. Within the HDL fraction, however, the larger HDL₂ subfractions were significantly elevated (p<0.002), and the smaller, more dense HDL₃ was lowered (p<0.002) by the salmon diet. These significant changes were detected by analytic ultracentrifugation and confirmed by gradient gel electrophoresis. Analysis of the apolipoproteins (apo) AI, AII, B, and E, and Lp(a) indicated only significant lowering of apoAI, consistent with the increased HDL₂, which is higher in cholesterol but lower in the major HDL apolipoprotein, apoAI. Thus, the purported beneficial cardiovascular effects of consumption of n−3 fatty acids by humans may, in part, be attributable to changes in the HDL distribution,i.e., the lowering of the more dense HDL₃ and the elevation of the larger, less dense HDL₂.     

Intercorrelations among plasma high density lipoprotein,obesity and triglycerides in a normal population

The interrelationships among fatness measures, plasma triglycerides and high density lipoproteins (HDL) were examined in 131 normal adult subjects: 38 men aged 27–46, 40 men aged 47–66, 29 women aged 27–46 and 24 women aged 47–66. None of the women were taking estrogens or oral contraceptive medication. The HDL concentration was subdivided into HDL_2b, HDL_2a and HDL₃ by a computerized fitting of the total schlieren pattern to reference schlieren patterns. Anthropometric measures employed included skinfolds at 3 sites, 2 weight/height indices and 2 girth measurements. A high correlation was found among the various fatness measures. These measures were negatively correlated with total HDL, reflecting the negative correlation between fatness measures and HDL₂ (as the sum of HDL_2a and_2b). Fatness measures showed no relationship to HDL₃. There was also an inverse correlation between triglyceride concentration and HDL₂. No particular fatness measure was better than any other for demonstrating the inverse correlation with HDL but multiple correlations using all of the measures of obesity improved the correlations. Partial correlations controlling for fatness did not reduce any of the significant correlations between triglycerides and HDL₂ to insignificance. The weak correlation between fatness and triglycerides was reduced to insignificance when controlled for HDL₂. Presented (in part) at the Annual Meeting of the Oil Chemists' Society in St. Louis, MO, May 1978.     

Effect of essential fatty acid deficiency on the size and distribution of rat plasma HDL

Rat plasma high density lipoproteins (HDL) are comprised of two major particle size subpopulations, HDL₁ (255 Å?140 Å) and HDL₂ (140 Å?84 Å), in which the proportion of arachidonate in fatty acids of cholesteryl esters is greater than 50%. To determine whether decreased availability of arachidonate for cholesterol esterification would alter the distribution and/or amounts of the HDL subpopulations, we compared HDL subpopulations in EFA-deficient and control rats. To separate the effects of EFA deficiency and fat deficiency and to evaluate effects of different saturated fats, we used EFA-deficient diets that were fat-free or that contained 5% saturated fat. The control diets were the EFA-deficient diets plus 1% safflower oil. The saturated fats were hydrogenated coconut oil, hydrogenated cottonseed oil and saturated medium-chain triglycerides. All EFA-deficient diets decreased the proportion of the HDL₁ subpopulation and the peak diameter of the HDL₂ subpopulation. These changes appeared after quite brief EFA depletion in young rats and may be related to the increased liver cholesteryl ester concentrations typical of EFA-deficient rats.     

The distribution of serum high density lipoprotein subfractions in non-human primates

The ultracentrifugal flotation patterns in 1.2 g/ml solvent and ultracentrifugal gradient distribution of high density lipoproteins (HDL) from the primates-human, apes and monkeys-were determined, with emphasis on the gorilla species of apes and rhesus monkeys. Diets for non-human primates were commercial chow, which is low in cholesterol. Molecular weights and protein, cholesterol, phospholipid and triglyceride compositions of various density fractions were determined on human, gorilla and rhesus HDL. The HDL₂/HDL₃ ratio was determined from the two peaks observed upon flotation in high salt in the analytical ultracentrifuge. The HDL₂ of all three species of apes-gorillas (Gorilla gorilla), chimpanzees (Pan troglodytes) and orangutans (Pongo pygmaeus)—was always greater than HDL₃, while that of all six species of Old World monkeys-Rhesus (Macaca mulatta), sooty mangabeys (Cercocebus atys), cynomolgus (Macaca fascicularis), stumptails, (Macaca arctoides) patas (Erythrocebus patas) and African greens (Cercopithecus aethiops)—was less. In addition, the HDL₃ concentration in five gorillas was about 15 mg/dl as cholesterol while the HDL₂ concentration was 92 mg/dl, much lower and higher, respectively, than humans. HDL₂ of gorillas was similar in density and molecular weight to that of humans. The distribution of densities in gorilla HDL was predominantly in HDL₂, while rhesus HDL usually, but not always, was unimodal, having a density distribution similar in heterogeneity to human HDL₃, but somewhat less dense (peaking at 1.109 vs 1.129 g/ml). The molecular weight of rhesus HDL was about the same as human HDL₃ in all three density subfractions and at the peak density. Likewise, the chemical compositions were similar for the subfractions 1.10–1.125 and>1.125 g/ml for rhesus HDL and human HDL₃. Consequently most but not all chow-fed rhesus HDL was very similar to human HDL₃, but lighter in density. A preliminary report of this study was given at the American Society for Biological Chemists Meeting in New Oreleans in April 1982.     

Human low density lipoprotein structure: Correlations with serum lipoprotein concentrations

Human low density lipoproteins (LDL) were isolated and purified from individuals having widely differing serum lipid concentrations. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) were also isolated and quantitated. HDL₂ and HDL₃ were separated by flotation velocity in the analytical ultracentrifuge and their relative weight percent determined. The mean density of LDL from 41 individuals was determined by flotation velocity at two different solvent densities. The mean density of LDL was directly proportional to the triglyceride (r=0.65) and VLDL (r=0.50) concentrations and inversely proportional to the HDL (r=−0.55) and HDL₂ (r=−0.74) concentrations (all significant at P<0.001). The mean molecular weight of LDL from 42 individuals was determined by flotation equilibrium centrifugation. The mean molecular weight of LDL was directly proportional to the HDL (r=0.49) and HDL₂ (r=0.48) concentrations and inversely proportional to the serum triglyceride (r=−0.60) and VLDL (r=−0.48) concentrations (all significant at P<0.005 except triglyceride—P<0.001). The molecular weight of LDL was inversely proportional to its density, and thus inversely proportional to its protein/lipid ratio which was confirmed by composition measurements. The density and molecular weight of LDL had no relationship to the concentration of LDL (r=0.04 and 0.03). A preliminary report of this study was given at the American Society for Biological Chemists Meeting in St. Louis, June 1981.     

Plasma high density lipoprotein subgroup distribution in rats fed diets with varying amounts of sucrose and sunflower oil

The effect of varying the dietary sunflower oil/sucrose (SO/SU) ratio on rat plasma lipid concentration and lipoprotein distribution was studied. Four groups of 10 rats were fed for 4 weeks diets with varying SO/SU ratios. Lipoprotein components were then estimated in whole plasma and after cumulative density ultracentrifugation. Whole plasma triacylglycerol (TG), total cholesterol (TC) and free cholesterol (FC) decreased with increasing SO/SU ratio; the CE/FC ratio increased, because CE remained virtually unaltered. Plasma TG-lowering was due to a decrease in VLDL and LDL-TG. Protein, CE and FC in d=1.063–1.100 g/ml (HDL_2b) and d=1.100–1.125 g/ml (HDL_2a) lipoproteins decreased upon increasing the SO/SU ratio. In contrast, in d=1.125–1.200 g/ml (HDL₃) lipoproteins, there was a concomitant increase in these components. Although increasing the SO/SU ratio effected more protein and CE transportation in HDL₃ and less in HDL₂, the total amount of these components in high density lipoproteins (d=1.063–1.200 g/ml) remained constant. Apo A-I and apo C-III decreased in HDL₂ but increased in HDL₃ upon increasing the SO/SU ratio. Also, HDL₂ apo E, and the apo C-II/apo C-III and small apo B/large apo B ratios in VLDL and LDL were lowered by increasing the SO/SU ratio. The hepatic VLDL-TG output during isolated liver perfusion was lowest in rats fed the diet with the highest SO/SU ratio. In perfusate, like in plasma, the VLDL and LDL apo C-II/apo C-III ratio, as well as the small apo B/large apo B ratio, decreased upon increasing the dietary SO/SU ratio. The results indicate that there can be appreciable diet-dependent variations in plasma HDL subgroup distribution in spite of unchanged total HDL levels.     

15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells

Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL₃ on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL₃ failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.     

Plasma,apolipoprotein A-I and A-II levels in hyperlipidemia

Some of the component moieties of high denisty lipoproteins (HDL) were analyzed in normal subjects and in patients with hyperlipidemia. Apoproteins A-I and A-II were quantified by radioimmunoassay, HDL cholesterol and triglycerides were assessed on heparin-MnCl₂ supernates of fasting plasmas. We found that HDL is enriched in triglycerides in all forms of hyperlipidemia, while the proportion of ApoA-II is unaltered and the proportion of ApoA-I is decreased. Thus, the composition of HDL is altered in hypertriglyceridemia. The molecular associations of ApoA-I and ApoA-II in plasma were also examined by assaying the apoprotein contents of plasma fractions prepared by ultracentrifugation and by gel filtration column chromatography. The ApoA-I contents of d<1.063 fraction increased in hyperlipidemia from <0.5% to ∼2%, but the ApoA-I contents of the d>1.21 fraction remained at <12% of total in plasmas with triglyceride levels <1500 mg/dl. d>1.21 ApoA-I rose to 23% in one plasma with a triglyceride level of >1700 mg/dl. On column chromatography, ApoA-I eluted with the lipoproteins and also in a fraction whose molecular weight (MW) appreared to be ∼50,000 daltons. The proportion of plasma ApoA-I which eluted in the 50,000 MW peak was positively correlated with plasma triglyceride levels, but at triglyceride levels of <1500 mg/dl, <20% of ApoA-I was in the 50,000 MW peak. Between levels of ∼2000 and 12,000 mg/dl, the percentage “50,000 M.W. ApoA-I” was 20–25%. The ApoA-II contents of d<1.063 fractions were also increased in hyperlipidemia, but >95% of ApoA-II was found in the HDL fractions in both normal and hyperlipidemic plasma both by column chromatography and ultracentrifugation. Thus, the molecular association of ApoA-I appears to be altered in hyperlipidemia.     

Influence of dietary egg and soybean phospholipids and triacylglycerols on human serum lipoproteins

Human serum lipid and lipoprotein concentrations and compositions were compared in ten healthy middle-aged men consuming phospholipids from egg or from soybean or triacylglycerol mixtures with fatty acid compositions similar to those of the phospholipids. All subjects followed each of the four treatments: egg phospholipids (EP), soybean phospholipids (SP), an oil of fatty acid composition similar to that of EP, and an oil similar in fatty acid composition to SP for six weeks with “wash-out” periods of similar duration between treatment periods. The phospholipids, 15 g/d, and the oils, 12 g/d, which contained approximately equivalent quantities of fatty acids were provided to the subjects in gelatin capsules and were taken before meals. Diet intake was monitored by three-day food records. Serum lipoproteins (L_p) were separated by ultracentrifugation into very low density lipoproteins, low density lipoproteins (LDL), high density lipoproteins (HDL)₂ and HDL₃. L_p fractions and whole serum were analyzed for triacylglycerols, cholesterol (CH), phospholipids (PL), and protein. HDL cholesterol was determined in while serum. Cholesteryl esters were determined in some L_p fractions. Lipid compositions of L_p were expressed in mmol/g protein. Apoprotein B was measured in whole serum and in LDL; apoprotein A-I in whole serum and in HDL₃. In whole serum, CH and PL were significantly lower after the SP compared to EP treatment periods. CH, but not PL, was lower after SPTG compared to EP. CH in HDL₂ was significantly higher after SP compared to SPTG. Also, PL in HDL₂ were significantly higher after SP compared to all other treatments and to baseline. Although human serum lipid responses to dietary phospholipids were generally the same as responses to ingested oils of comparable fatty acid composition, the data suggest the possibility that SP selectively increase HDL₂ cholesterol and phospholipids.     

Experimental nephrotic syndrome in the rat induced by puromycin aminonucleoside: Hepatic synthesis of lipoproteins and apolipoproteins

Hepatic synthesis of lipoproteins and apolipoproteins was investigated in male Wistar rats with severe nephrotic syndrome induced by puromycin aminonucleoside by incubating liver slices with a mixture of¹⁴C-amino acids. Labeled lipoproteins were separated by preparative ultracentrifugation from the incubation medium after the addition of carrier plasma. The incorporation of¹⁴C-amino acids into very low density lipoproteins (VLDL) (1.006 g/ml), low density lipoproteins (LDL) (1.006–1.063 g/ml) and high density lipoproteins (HDL) (1.063–1.210 g/ml) was increased in nephrotic liver 6.1-, 5.7- and 5.0-fold, respectively. The measurement of radioactivity associated to apolipoproteins isolated by SDS-PAGE documented an increased incorporation into apolipoprotein E (apoE) of nephrotic VLDL (33.1% vs 20% of the total radioactivity incorporated into VLDL apoproteins) and a markedly increased incorporation into apolipoprotein A-I (apoA-I) of nephrotic HDL (44.3% vs 16.3% of the total radioactivity incorporated into HDL apoproteins). In nephrotic liver, the total incorporation of amino acids into apolipoproteins (apoVLDL+apoLDL+apoHDL) was increased 12.6 times for apoA-I, 6.4 times for apoB, 5.0 times for apoE, 4.2 times for apoC+apoA-II and 2.5 times for apoA-IV. We suggest that, in nephrotic liver: (a) the synthesis of VLDL, LDL and HDL is increased, and (b) the total synthesis of apoA-I is selectively increased when compared to that of the other apolipoproteins. Preliminary reports of this work were presented at the Annual Meeting of the European Society for the Study of the Liver (Düsseldorf, September 13–15, 1979); at the 5th International Symposium on Atherosclerosis (Houston, November 6–9, 1979) and at the Annual Meeting of the Italian Society for the Study of the Liver (Rome, December 14–15, 1979).     

Interaction of discoidal complexes of dimyristoyl phosphatidylcholine-cholesterol-apolipoprotein A-I with human plasma high density lipoprotein HDL3

The interaction of human plasma high density lipoproteins (HDL₃) with discoidal complexes of apolipoprotein A-I (apoA-I) and dimyristoyl phosphatidylcholine (DMPC) containing 0, 10, 20 or 30 mol % cholesterol was investigated. Discoidal complexes containing various amounts of cholesterol were prepared by incubating apoA-I and DMPC-cholesterol liposomes for 12 hr at 25 C; the protein-lipid complexes were isolated by gel filtration chromatography on Bio-Gel A15m. Increasing the cholesterol content from 0 to 30 mol % caused a decrease in the fluidity of the discoidal complexes as determined by fluorescence polarization with 1,6-diphenyl-1,3,5-hexatriene; a reduced phase-transition amplitude; a decrease in the ratio of apoA-I to DMPC; and an increase in the width of the discoidal complexes as determined by electron microscopy after negative staining. Incubation of the apoA-I-lipid complexes with HDL₃ resulted in a complete breakdown of the discoidal structures and a transfer of DMPC and cholesterol to HDL₃. As a result of lipid transfer, there was an increase in the size of HDL₃. These in vitro results may be of significance as they relate to the interconversion of HDL subfractions during lipoprotein-lipase-induced lipolysis of triglyceride-rich lipoproteins.