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.     

Effect of chronic glucagon administration on lipoprotein composition in normally fed,fasted and cholesterol-fed rats

Male adult Wistar rats received daily (at 9 a.m. and 5 p.m.) 10 μg of zinc-protamine glucagon by subcutaneous injection for 8 days. Plasma cholesterol levels were decreased by 36% in fed rats, 33% in cholesterol-fed rats and by 55% in fasted rats. Lipoproteins were separated into 22 fractions by ultracentrifugation using a density gradient. Glucagon administration decreased the cholesterol content in all lipoproteins except low density lipoprotein (LDL₁) (1.006–1.040) and very low density lipoprotein (VLDL) from cholesterol-fed rats. The main decrease (−57 to −81%) was observed in 1.050–1.100 g/mL lipoproteins (LDL₂ and HDL₂), which contained a large amount of apo E, while HDL₃ cholesterol was not affected. Triacylglycerol levels were decreased only in chylomicrons and VLDL (−70%) of fed and cholesterol-fed rats, while plasma and lipoprotein triacylglycerol levels were not changed in fasted rats treated with glucagon. In normally fed rats glucagon administration increased by 42% the fractional catabolic rate of [¹²⁵I]HDL₂ while the absolute catabolic rate appeared to be unchanged. Glucagon seems to be a potent hypolipidemic agent affecting mainly the apo E-rich lipoproteins. Its chronic administration limits lipoprotein accumulation which occurs upon cholesterol feeding.     

Determination of HDL2 cholesterol by precipitation with dextran sulfate and magnesium chloride: Establishing optimal conditions for rat plasma

Optimal conditions for analyzing HDL₂ cholesterol in small amounts of rat plasma have been studied using different concentrations of dextran sulfate and MgCl₂ to precipitate lipoproteins containing apolipoprotein B and/or apo E. When the MgCl₂ level was 91 mM, the supernate cholesterol was rather constant at a level of about 50–60% of the total plasma cholesterol concentration. Immunochemical determination of the apo A-I content indicated that no major losses of the HDL₂ fraction took place under these conditions. The recovery of about 96% of HDL₂ lipoproteins after the precipitation of rat plasma and the almost complete absence of lipoproteins belonging to the VLDL, LDL and HDL₁ fractions was demonstrated by agarose gel electrophoresis. Thus, the method should be suitable for screening the HDL₂ cholesterol content in small volumes of rat plasma.     

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.     

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.     

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.     

Induction of long-lasting hypercholesterolemia in the rat fed a cystine-enriched diet

The influence of dietary excess (5%) of L-cystine on rat plasma lipoproteins was examined. After only one week of cystine feeding, an increase in the plasma cholesterol level and a decrease in triglyceride levels were observed. The increase in cholesterol level became greater when the duration of cystine-enriched diet increased until eight weeks (+131% after eight weeks), but no further increase occurred between 8 and 20 weeks. This change was essentially due to the progressive increase in cholesterol levels in high density lipoproteins (HDL) and in lipoproteins isolated between 1.040 and 1.063 g/ml, i.e., certain low density lipoproteins (HDL₂), and containing mainly apoE-rich lipoproteins (HDL₁). The decrease in plasma triglycerides resulted from that of chylomicrons and very low density lipoproteins (VLDL). The effects observed after four or eight weeks of cystine feeding were maintained for eight weeks after replacing the cystine diet by the standard diet. Ingestion of the standard diet containing either cholestyramine (2%) or probucol (0.25%) following eight weeks of cystine feeding significantly decreased plasma cholesterol levels. It is concluded that cystine-fed rats are a useful tool of investigation for understanding mechanisms leading to increased plasma cholesterol level and for hypocholesterolemic drug trials.     

Apolipoprotein A-I,A-IV and E synthesis in the liver of copper-deficient rats

Copper deficiency induces hypercholesterolemia in the rat. This hypercholesterolemia is mainly due to an increase in apo E-rich high density lipoproteins (HDL₁). The present study was undertaken to determine whether the HDL increase could be explained by altered low-molecular weight apolipoprotein (apo) synthesis in the liver. The effect of copper deficiency on apo A-I, apo A-IV and apo E concentrations in plasma, as well as on respective mRNA levels and synthesis in the liver, were therefore investigated. We observed that the increased HDL₁ levels in the plasma of copper-deficient rats were associated with a significant rise in plasma apo E concentrations; however, plasma apo A-I and apo A-IV concentrations remained unchanged. Liver apo synthesis and respective apo mRNA levels were not significantly altered in copper-deficient animals when compared to control rats. No changes in apo E mRNA levels in various tissues from copper-deficient, as compared to control rats, were noted. Based on the data obtained, it was concluded that the observed changes in plasma lipoprotein and apo concentrations are not related to changes in low-molecular weight apo synthesis in the liver. The mechanisms of the impaired catabolism of HDL₁ should be further evaluated to possibly explain the observed increase in this fraction in copper-deficient rats.     

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.     

The role of high density lipoprotein apolipoprotein CII in triglyceride metabolism

The purpose of these studies was (a) to examine the relationship between total plasma triglycerides (TG) and the amount of apolipoprotein CII (apo CII) in triglyceride rich lipoproteins (TRL), and (b) to determine whether TRL could be enriched with apo CII in vitro. In 13 patients with primary endogenous hypertriglyceridemia, (log₁₀) total plasma TG correlated inversely with the amount of apo CII per unit very low density lipoprotein (VLDL) protein (r=−0.76;p<0.005) and VLDL TG (r=−0.75; p<0.005). The potency of VLDL to activate milk lipoprotein lipase (LPL) in hydrolyzing triolein was studied in vitro. LPL activator potency per unit VLDL protein or VLDL TG correlated inversely with (log₁₀) total plasma TG (r=−0.86 and r=−0.76, respectively; p<0.005). LPL activator potency per nM VLDL apo CII also correlated inversely with (log₁₀) total plasma TG (r=−0.49; p<0.01). In seven patients with familial type V hyperlipoproteinemia, the average amount of apo CII in TRL protein was subnormal (5.86±0.62% vs 10.0±0.51% in normal subjects). The higher the (log₁₀) total plasma TG, the lower was the apo CII content in TRL protein (r=−0.93; p<0.01). To determine the factors governing the distribution of apo CII between lipoproteins and whether TRL could be enriched with apo CII, five approaches were undertaken: (a)¹²⁵I apo CII was added to mixtures of VLDL and HDL. The amount of labelled apo CII in VLDL was proportional to the ratio of VLDL to HDL. (b) TRL from four patients with familial type V hyperlipoproteinemia was incubated with high density lipoprotein (HDL) from a normal subject. An increase in the TRL/HDL ratio was associated with transfer of apo CII from HDL to TRL and a reciprocal transfer of non-apo CII protein from TRL to HDL. Net apo CII enrichment of TRL protein was possible below a HDL/TRL protein ratio of ca. 6 under the experimental conditions. (c) A fixed amount of normal plasma feed of TRL was incubated with different amounts of TRL from two patients with familial type V hyperlipoproteinemia. The amount of apo CII that transferred from normal TRL free plasma to the patient’s TRL was proportional to the amount of TRL in the mixture. (d) A doubling and tripling in the amount of apo CII in TRL was found when apo CII was added directly to TRL from a normal subject and TRL from a patient with familial type V hyperlipoproteinemia, respectively. (e) When apo CII was added directly to normal plasma and plasma from a patient with primary type IV hyperlipoproteinemia, the peptide was taken up mainly by VLDL and HDL, indicating enrichment of these fractions. The distribution of the added apo CII in each lipoprotein fraction resembled the distribution in the native plasma. TRL was isolated after addition of apo CII to plasma from two patients with familial types IV and V, respectively. Enrichment of TRL with apo CII was associated with an approximate 1.5-fold increase in the LPL activator potency per unit TRL protein. These studies suggest that firstly, the amount of apo CII in TRL is inversely related to the severity of hypertriglyceridemia. Secondly, the distribution of apo CII between TRL and HDL is governed by the mass ratios of these two lipoprotein classes. Thirdly, plasma TRL and HDL have a reserve binding capacity of apo CII and fourthly, it is possible to enrich these lipoproteins with this functionally important peptide. Whether net enrichment of TRL with apo CII and also an increase in its biological activity to activate LPL in vitro is related to increased in vivo catabolic rate requires to be determined.     

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 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.     

Activation of the phospholipase A1 activity of lipoprotein lipase by apoprotein C-II

The effect of apo very low density lipoprotein (apo VLDL) and apoprotein C-II on the phospholipase A₁ activity associated with lipoprotein lipase (E.C.3.1.1.3) was studied using purified bovine milk lipoprotein lipase. The enzyme degraded¹⁴C phosphatidylcholine (PC) to¹⁴C 2-acyl lysophosphatidylcholine at a rate of 0.28±0.01 nmol/min/ml and triolein at a rate of 20.3±0.4 nmol/min/ml in mixed emulsions of PC and triolein. The phospholipase activity and triacylglycerol lipase activity were both increased by the addition of apo VLDL and apoprotein C-II. After maximal activation, the rate of PC degradation was 1.19±0.02 nmol/min/ml and triolein degradation 64.4±0.4 nmol/min/ml. Activation of phospholipase A₁ activity and triacylglycerol lipase activity occurred in parallel.     

Plasma lipoproteins in dairy cows with naturally occurring severe fatty liver: Evidence of alteration in the distribution of apo A-I-containing lipoproteins

The relationships between fatty liver in dairy cows and reduced levels of plasma lipoproteins, and particularly of low density lipoproteins (LDL), has been previously described. Since electrophoretic heterogeneity of ultracentrifugally isolated LDL (d, 1.006–1.063 g/ml) has been found, the exact nature of this reduction in cows with fatty liver was investigated. Lipoproteins from control and severely afflicted animals were isolated by ultracentrifugation and affinity chromatography on heparin-Sepharose CL 6 B. Gradient gel electrophoresis of lipoproteins on 4–30% gels and an immunolocalization study of apoprotein A-I (apo A-I) showed that control animals have two subpopulations of apo A-I-containing particles with a mean radius of 6.52 and 5.05 nm. In the fatty liver cows, the former was clearly shifted toward smaller particles. We concluded that the depressed level and compositional modifications of LDL in severe fatty liver cows result from a decrease in the oversized apo A-I-containing lipoproteins which can be isolated in the LDL density range. This could stem from the decreased supply of triglyceride-rich lipoprotein surface components for the production of these lipoproteins. The modifications can be plausibly explained by a reduced synthesis or secretion of very low density lipoproteins (VLDL) by the liver.     

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.     

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.     

Carbohydrate type and amount alter intravascular processing and catabolism of plasma lipoproteins in guinea pigs

To test the effects of exchanging dietary complex and simple carbohydrate for fat calories on lipoprotein metabolism, guinea pigs were fed two different fat/carbohydrate ratios: 2.5∶58% (w/w) or 25∶29% (w/w) with either sucrose or starch as the carbohydrate source. Animals fed high-fat had higher plasma low-density lipoprotein (LDL) and hepatic cholesterol concentrations than animals fed low-fat diets (P<0.01). The cholesteryl ester content per particle was higher, and the number of triacylglycerol (TAG) molecules was lower in very low density lipoprotein (VLDL) and LDL from animals fed high-fat diets. Intake of high-fat/sucrose resulted in higher plasma LDL concentrations than intake of high-fat/starch, and animals fed low-fat/starch had the highest plasma TAG concentrations associated with VLDL particles containing more TAG molecules, as well as a TAG-enriched LDL. The activity of plasma lecithin cholesteryl:acyl transferase (LCAT) was highest in animals fed high-fat/sucrose, and heart lipoprotein lipase (LPL) activity was higher in animals fed high-fat diets. Hepatic apoprotein B/E (apo B/E) receptor number (B_max) was increased 21% with low-fat diets (P<0.01). These results suggest that the hypercholesterolemia induced by high-fat and by sucrose intake are associated with a higher plasma LCAT activity which results in a cholesteryl ester-enriched VLDL which, by the action of LPL, might be more readily converted to LDL through the delipidation cascade leading to downregulation of hepatic apo B/E receptors. The hypertriglyceridemia associated with low-fat intake may result from increased production of VLDL TAG, which would explain the increased TAG content and the higher TAG/CE ratio of VLDL from animals fed the low-fat/starch diet.     

Diet-induced type IV-like hyperlipidemia and increased body weight are associated with cholesterol gallstones in hamsters

Male Syrian hamsters (60–70 g) were fed purified diets containing 5% fat (American Fat Blend) and 15% fiber with or without 0.3% cholesterol (0.86 mg/kcal), for 12 weeks. Hamsters fed the cholesterol-supplemented challenge diet revealed a major increase in plasma triglyceride between 9 and 12 weeks, whereas plasma cholesterol (which reflected body weight dynamics) increased three-fold up to nine weeks and plateaued (342±22vs. 122±5 mg/dL). The greatest increases in cholesterol occurred in the very low density lipoprotein (VLDL) and high density lipoprotein (HDL₂) fractions. Gallstone incidence was similar (69%vs. 78%) for cholesterol-supplementedvs. control hamsters, but the type of stones differed. Of the cholesterol-supplemented hamsters with gallstones, 45% had cholesterol stones and 55% had pigment stones. Only pigment stones were seen in control hamsters. Hamsters with cholesterol stones were 25% heavier and transported most cholesterol in VLDL (33±5%), approximately double that in VLDL of cholesterol-supplemented hamsters with no stones (19±3%) or cholesterol-supplemented hamsters with pigment stones (21±3%). Hamsters with pigment stones or no stones (regardless of diet fed) transported the majority of their cholesterol in HDL₂ (44%), whereas this figure was only 27% in hamsters that developed cholesterol stones. Thus pigment stones develop routinely in hamsters fed casein-based purified diets. Adding dietary cholesterol resulted in cholesterol gallstones only in those hamsters that gained the most weight and whose terminal VLDL/HDL cholesterol ratio exceeded 1.0, not unlike the lipoprotein profile of obese humans who develop cholesterol gallstones. Presented in part at the Xth International Symposium on Drugs Affecting Lipid Metabolism, November 8–11, 1989, Houston, TX.     

Separation of the apoprotein components of human very low density lipoproteins by ion-paired,reversed-phase high performance liquid chromatography

A number of crude apolipoprotein samples isolated from human very low density lipoproteins (VLDL) were analyzed by reversed phase high performance liquid chromatography. The mobile phase consisted of a 1% solution of the polar ion-pairing reagent triethylammonium phosphate. A slow, nonlinear gradient of acetonitrile (37–42%) was used to elute the apolipoproteins. The order of elution was as follows: apolipoprotein C_x, apolipoprotein C-I, apolipoprotein C-III₂, apolipoprotein C-III₁, apolipoprotein C-III₀ and apolipoprotein C-II. This order is consistent with the known polarity of the proteins, i.e., the most nonpolar, apolipoprotein C-II, was the last to be eluted, whereas apolipoprotein C-I, with the lowest nonpolar surface area eluted first. The recovery of the individual apolipoproteins was 80–95% and the individual peaks were characterized by amino acid analysis, UV absorption spectra and chromatography of pure protein standards.