Fluorescence-based single-strand conformation polymorphism analysis of the low density lipoprotein receptor gene by capillary electrophoresis

We describe here a new method to screen for unknown mutations in the low density lipoprotein (LDL) receptor gene by the use of capillary electrophoresis in single-strand conformation polymorphism (SSCP) analysis. To analyze the promoter and all 18 exons, 20 different amplification reactions were necessary. For each polymerase chain reaction (PCR), the forward and reverse primers were 5′ fluorescent-labelled with FAM and HEX, respectively. To test the accuracy of the newly developed method, 61 genetic variants distributed in 16 exons were analyzed. Under identical electrophoresis conditions (13 kV, 30°C, 30 min), 59 mutations were detected by a distinct abnormal SSCP pattern. The two remaining mutations showed only slight abnormalities, which could be amplified by increasing the electrophoresis temperature. The high accuracy, the degree of automation and the speed of analysis make fluorescence-based SSCP analysis with capillary electrophoresis ideal for rapid mutation screening and the technique is well-suited for clinical applications.     

Development of an immunoblot assay with infrared fluorescence to quantify paraoxonase 1 in serum and plasma

Paraoxonase 1 (PON1) requires calcium for activity and is inactivated in the presence of EDTA. Because of this, studies to date have used serum or heparinized plasma for both activity and mass assays of PON1. Whole serum and EDTA plasma were analyzed by SDS-electrophoresis and Western blot using anti-PON1 monoclonal antibody 4C10. Because PON1 has one disulfide and one free cysteine residue, the samples were reduced with dithiothreitol before electrophoresis. Western blot identified a major PON1 band with a molecular mass of approximately 45 kDa and two minor bands of approximately 40 and 35 kDa in both serum and EDTA plasma. This established that PON1 is inactive, but structurally intact, in EDTA plasma and suggested that a mass assay could be developed based on SDS-electrophoresis and Western blot. Linearity was established for plasma and for a PON1 standard. Quantification was based on the major PON1 band at 45 kDa. The correlation between serum and plasma PON1 mass was 0.9553. The between-run variation was determined with a serum pool to be 7.8%. The mass of PON1 in serum was significantly correlated with arylesterase activity (r = 0.85). Thus, we have demonstrated the feasibility of measuring PON1 mass in either serum or EDTA plasma.     

Effects of emulsified policosanols with different chain lengths on cholesterol metabolism in heterozygous LDL receptor-deficient mice

Policosanol is a mixture of long-chain primary aliphatic saturated alcohols. Previous studies in humans and animals have shown that these compounds improved lipoprotein profiles. However, more-recent placebo-controlled studies could not confirm these promising effects. Octacosanol (C28), the main component of sugarcane-derived policosanol, is assumed to be the bioactive component. This has, however, never been tested in an in vivo study that compared individual policosanol components side by side. Here we present that neither the individual policosanol components (C24, C26, C28, or C30) nor the natural policosanol mixture (all 30 mg/100 g diet) lowered serum cholesterol concentrations in LDL receptor knock-out (LDLr(+/-)) mice. Moreover, there was no effect on gene expression profiles of LDLr, ABCA1, HMG-CoA synthase 1, and apolipoprotein A-I (apoA-I) in hepatic and small intestinal tissue of female LDLr(+/-) mice after the 7 week intervention period. Finally, none of the individual policosanols or their respective long-chain fatty acids or aldehydes affected de novo apoA-I protein production in vitro in HepG2 and CaCo-2 cells. Therefore, we conclude that the evaluated individual policosanols, as well as the natural policosanol mixture, have no potential for reducing coronary heart disease risk through effects on serum lipoprotein concentrations.     

Simultaneous determination of molecular species of monoacylglycerols,diacylglycerols and triacylglycerols in human very-low-density lipoproteins by reversed-phase liquid chromatography

The aim of the present study was to investigate the applicability of a previously developed method for the analysis of triacylglycerol molecular species to the simultaneous determination of triacylglycerols, diacylglycerols and monoacylglycerols of human very-low-density lipoproteins (VLDL). Ten elderly women were recruited for the study. Blood was obtained in fasting conditions and VLDL were isolated by ultracentrifugation. Neutral lipids were separated by solid-phase extraction and were subsequently injected on a reversed-phase HPLC system, with an elution system composed of acetone in acetonitrile. The method allowed the separation of four monoacylglycerols, 18 diacylglycerols and 24 triacylglycerols, including the resolution of positional isomers of diacylglycerols. Monoacylglycerols were composed of oleic, linoleic, palmitic and stearic acids. The major diacylglycerols were 1,2-dilinoleoyl-glycerol and 1,3-dilinoleoyl-glycerol (14.24+/-1.02 and 17.93+/-1.42%, respectively). The main triacylglycerols quantified were dioleoyl-stearoyl-glycerol (OOS), oleoyl-dipalmitoyl-glycerol (OPP), trilinoleoyl-glycerol (LLL) and linoleoyl-distearoyl-glycerol (LSS), accounting for 11.25+/-2.15, 10.14+/-2.05, 9.35+/-2.30 and 8.56+/-1.56%, respectively. An inverse relationship between polarity and fatty acid disappearance from triacylglycerols (r(2)=0.82, P<0.05) and from diacylglycerols (r(2)=0.93, P<0.01) was discovered. In conclusion, the method allowed, for the first time, the easy, rapid and simultaneous determination in a single chromatogram of triacylglycerol, diacylglycerol and monoacylglycerol molecular species of human VLDL by reversed-phase HPLC.     

Molecular characteristics of lipophorin,the juvenile hormone-binding protein in the hemolymph of the Colorado potato beetle

Lipophorin, the protein that specifically binds juvenile hormone in the hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, is a high-density lipoprotein of M_r ～ 574,000. Lipophorin contains 43% lipid and is composed of two apoproteins: apolipophorin I (M_r ～ 251,000) and apolipophorin II (M_r ～ 78,000). Both apoproteins contain mannose residues. Carotenoids make up a substantial part of the lipid fraction. Lipophorin constitutes about 25% of the total hemolymph proteins. Its concentration in the hemolymph (26 μM in 4-day-old long-day and 40 μM in 4-day-old short-day beetles) changes with different physiological conditions concomitant with changes in total protein content. Lipophorin specifically binds 10R-juvenile hormone III with high affinity. The dissociation constant for 10R-juvenile hormone III is 12 ± 2 nM. One lipophorin molecule contains one specific juvenile hormone-binding site. The concentration of binding sites therefore equals that of lipophorin in hemolymph.     

The National Niemann-Pick Type C1 Disease Database: correlation of lipid profiles, mutations, and biochemical phenotypes

Niemann-Pick type C1 disease (NPC1) is an autosomal recessive lysosomal storage disorder characterized by neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration. The present study provides the lipid profiles, mutations, and corresponding associations with the biochemical phenotype obtained from NPC1 patients who participated in the National NPC1 Disease Database. Lipid profiles were obtained from 34 patients (39%) in the survey and demonstrated significantly reduced plasma LDL cholesterol (LDL-C) and increased plasma triglycerides in the majority of patients. Reduced plasma HDL cholesterol (HDL-C) was the most consistent lipoprotein abnormality found in male and female NPC1 patients across age groups and occurred independent of changes in plasma triglycerides. A subset of 19 patients for whom the biochemical severity of known NPC1 mutations could be correlated with their lipid profile showed a strong inverse correlation between plasma HDL-C and severity of the biochemical phenotype. Gene mutations were available for 52 patients (59%) in the survey, including 52 different mutations and five novel mutations (Y628C, P887L, I923V, A1151T, and 3741_3744delACTC). Together, these findings provide novel information regarding the plasma lipoprotein changes and mutations in NPC1 disease, and suggest plasma HDL-C represents a potential biomarker of NPC1 disease severity.     

Effects of the cholesteryl ester transfer protein inhibitor torcetrapib on VLDL apolipoprotein E metabolism

Cholesteryl ester transfer protein (CETP) inhibition leads to changes in lipoprotein metabolism. We studied the effect of the CETP inhibitor torcetrapib on VLDL apolipoprotein E (apoE) metabolism. Subjects, pretreated with atorvastatin (n = 9) or untreated (n = 10), received placebo followed by torcetrapib (4 weeks each). After each treatment, subjects underwent a primed-constant infusion of D(3)-leucine to determine the VLDL apoE production rate (PR) and fractional catabolic rate (FCR). Torcetrapib alone reduced the VLDL apoE pool size (PS) (-28%) by increasing the VLDL apoE FCR (77%) and leaving the VLDL apoE PR unchanged. In subjects pretreated with atorvastatin, torcetrapib increased the VLDL apoE FCR (25%) and PR (21%). This left the VLDL apoE PS unchanged but increased the VLDL apoE content, likely enhancing VLDL clearance and reducing LDL production in this group. Used alone, torcetrapib reduces the VLDL apoE PS by increasing the apoE FCR while leaving the VLDL apoE content unchanged. In contrast, torcetrapib added to atorvastatin treatment increases both the VLDL apoE FCR and PR, leaving the VLDL apoE PS unchanged. Adding torcetrapib to atorvastatin treatment increases the VLDL apoE content, likely leading to decreased conversion of VLDL to LDL, reduced LDL production, and lower levels of circulating VLDL and LDL.     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp^55/57→Ala^55/57 substitution of kringle (K)IV₁₀)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV₁₀ LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp⁵⁷→Ala⁵⁷ substitution in KIV₁₀ that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV₁₀ LBS and is unique to human apo(a), which may explain Lp(a)’s pro-atherogenic potential.     

Effects of a 1,3-diacylglycerol oil-enriched diet on postprandial lipemia in people with insulin resistance

Postprandial hypertriglyceridemia is common in individuals with insulin resistance, and diets enriched in 1,3-diacylglycerol (DAG) may reduce postprandial plasma triglycerides (PPTGs). We enrolled 25 insulin-resistant, nondiabetic individuals in a double-blind, randomized crossover trial to test the acute and chronic effects of a DAG-enriched diet on PPTG. Participants received either DAG or triacylglycerol (TAG) oil, in food products, for 5 weeks. Fasting lipids, and two separate postprandial tests, one with DAG oil and one with TAG oil, were performed at the end of each 5 week diet period. We found no acute or chronic effects of DAG oil on PPTG. Thus, neither the DAG oil PPTG (h/mg/dl) on a chronic TAG diet [area under the curve (AUC) = 503 +/- 439] nor the TAG oil PPTG on a chronic DAG diet (AUC = 517 +/- 638) was different from the TAG oil PPTG on a chronic TAG diet (AUC = 565 +/- 362). Five weeks of a DAG-enriched diet had no acute or chronic effects on PPTG in insulin-resistant individuals. We suggest further studies to evaluate the effects of DAG on individuals with low and high TG levels.     

The Effect of Dehydroepiandrosterone Combined with a Low-Fat Diet in Spontaneously Obese Dogs: A Clinical Trial

Dehydroepiandrosterone (DHEA) has been shown to have antiobesity activity in rodents and spontaneously obese dogs. This study evaluated the effect of DHEA or placebo combined with a low-fat/high-fiber diet in spontaneously obese dogs in a clinical trial. Spontaneously obese, euthyroid dogs, referred to the University of Wisconsin School of Veterinary Medicine for treatment of their obesity, were evaluated for percent overweight, rate of weight loss, serum cholesterol, plasma lipoprotein and serum biochemistry profiles, complete blood count, and endocrine profiles (T4, T3, Cortisol, insulin, and DHEA-sulfate). DHEA-treated dogs had a significantly increased rate of actual and percent excess weight loss compared with placebo-treated dogs. Serum cholesterol decreased in both treatment groups; however, DHEA-treated dogs had a significantly greater reduction than placebo-treated dogs. DHEA-treated dogs had a significant 32% reduction in total plasma cholesterol, which was due to a 27% reduction in the lipoprotein fraction containing the high-density lipoprotein (HDL) and a 50% reduction in the lipoprotein fraction containing the low-density lipoprotein (LDL). Placebo-treated dogs did not have a significant reduction in total plasma cholesterol or in the fraction containing LDL; however, they did have a significant 11% reduction in the fraction containing HDL. Significant decreases in serum T4 and T3 observed in dogs receiving DHEA were not noted in dogs receiving placebo. DHEA in combination with caloric restriction results in a faster rate of weight loss than does caloric restriction alone. In addition, DHEA has hypocholesterolemic activity, particularly affecting the lipoprotein fraction containing the LDL cholesterol.     

Genetic aspects of susceptibility to obesity and related dyslipidemias

Obesity has a multifactorial origin. However, although environmental variables undoubtedly play a role in the development of obesity, it is now clear that genetic variation is also involved in the determination of an individual's susceptibility to body fat accumulation. In addition, it is also widely accepted that obesity is not a single homogeneous phenotype. It is also heterogeneous regarding its causes and metabolic complications. The regional distribution of body fat appears to be an important correlate of the metabolic complications that have been related to obesity. Due to their higher accumulation of abdominal fat, men are generally more at risk for the metabolic complications of obesity than women whereas some obese women, with large gluteal-femoral adipose depots may have a cosmetic problem which may not necessarily require medical intervention. Several studies have been conducted to understand the mechanisms by which abdominal obesity is related to diabetes, hypertension and cardiovascular disease. It appears that the increased risk of abdominal obesity is the result of complex hormonal and metabolic interactions. Studies in genetic epidemiology have shown that both total body fatness and the regional distribution of body fat have a significant genetic component. Standardized intervention studies using an identical twin design have shown that individuals that have the same genetic background tend to show similar changes in body fat and in plasma lipoprotein levels when exposed to standardized caloric excess or energy restriction. Finally, although abdominal obesity is a significant risk factor for cardiovascular disease, not every abdominal obese subject will experience metabolic complications, suggesting that some obese individuals may be more susceptible than others. Variation in several genes relevant to lipid and lipoprotein metabolism may alter the relation of abdominal obesity to dyslipoproteinemias. Abdominal obesity should therefore be considered as a factor that exacerbates an individual's susceptibility to cardiovascular disease.     

Origins of intestinal ABCA1-mediated HDL-cholesterol

The origins of cholesterol utilized by intestinal ABCA1 were investigated in the human intestinal cell line Caco-2. Influx of apical membrane cholesterol increases ABCA1 mRNA and mass, resulting in enhanced efflux of HDL-cholesterol. Luminal (micellar) cholesterol and newly synthesized cholesterol are not transported directly to ABCA1 but reach the ABCA1 pool after incorporation into the apical membrane. Depleting the apical or the basolateral membrane of cholesterol by cyclodextrin attenuates the amount of cholesterol transported by ABCA1 without altering ABCA1 expression. Filipin added to the apical side but not the basal side attenuates ABCA1-mediated cholesterol efflux, suggesting that apical membrane "microdomains," or rafts, supply cholesterol for HDL. Preventing cholesterol esterification increases the amount of cholesterol available for HDL. Ezetimibe, a Niemann-Pick C1-like 1 protein inhibitor, does not alter ABCA1-mediated cholesterol efflux. U18666A and imipramine, agents that mimic cholesterol trafficking defects of Neimann-Pick type C disease, attenuate cholesterol efflux without altering ABCA1 expression; thus, intestinal NPC1 may facilitate cholesterol movement to ABCA1. ABCA1-mediated cholesterol efflux is independent of cholesterol synthesis. The results suggest that following incorporation into plasma membrane and rafts of the apical membrane, dietary/biliary and newly synthesized cholesterol contribute to the ABCA1 pool and HDL-cholesterol. NPC1 may have a role in this process.     

High-Density Lipoprotein Aggregated by Oxidation Induces Degeneration of Neuronal Cells

Abstract: We have previously reported that high-density lipoprotein (HDL) exhibits antineuritogenic effects on chicken cerebral cells in culture. In the present study, we show the effects of HDLs, oxidized by UV irradiation or heating, on chicken cerebral neurons in culture. Both treatments produced several physical and chemical changes in the HDLs, i.e., formation of lipid peroxides, enlargement of HDL diameters, an increased exposure of the tryptophan groups of the apolipoprotein A-I to a more hydrophilic environment, formation of bityrosines, and cross-linking of apolipoprotein A-I. When these treatments were performed in the absence of EDTA, most of the modifications described above were more intense and HDLs formed a macroaggregate that displays a rosette-like structure. The aggregated HDLs produced neurodegeneration and death when added to both undifferentiated and differentiated cerebral neurons in culture. This process was accompanied by the disorganization of the cellular microtubular cytoskeleton and hyperphosphorylation of the microtubule-associated protein tau. Native HDL or HDLs treated in the presence of EDTA inhibited the neuritogenesis of undifferentiated neurons but did not show any significant effect on the differentiated neurons in culture. The effects on the cellular cytoskeleton and morphology of aggregated HDLs recall those of the fibrillar β-amyloid peptide. The present results suggest that aggregated HDLs could participate in neurodegeneration associated with oxidative stress in the CNS.     

The interaction of prostaglandins with human serum lipoproteins

Using high density and low density lipoproteins (HDL and LDL) labeled with fluorescent analogues of phosphatidylcholine or sphingomyelin it was found that low amounts (10^–12 M) of prostaglandins E₁ and F₂ induced different structural rearrangements of the lipoprotein surface, whereas prostaglandins E₂ and F₁ had no effect. The effects of prostaglandin E₁ on HDL were largely paralled by those of this prostaglandin on synthetic recombinants prepared from pure apolipoprotein A₁, phospholipids and cholesterol and were demonstrated to be caused by prostaglandin-apolipoprotein interaction. The interaction resembled that of a ligand with a specific receptor protein because it was specific, reversible, concentration and temperature dependent and saturable. However the retaining capacity of HDL or LDL for prostaglandin E₁ as determined by equilibrium dialysis was very low and a single prostaglandin E₁ molecule was able to induce structural changes in large numbers of discrete lipoprotein particles. To explain this remarkable fact a non-equilibrium model of ligand-receptor interaction is proposed. According to that model in open systems characterized by weak ligand-receptor binding, high diffusion rate of the ligand and long relaxation times which exceed the interval between two successive receptor occupations, the ligand-induced changes will accumulate, resulting in transformation of the system into a new state which may be far away from equilibrium. It is emphasized that the low mobility of lipids constituting the environment of the receptor protein plays a critcal role in this type of signal amplification.It was further demonstrated that the PGE₁-induced changes of the lipoprotein surface resulted in an enhancement of LDL-to-HDL transfer of cholesterol esters and phosphatidylcholine especially in the presence of serum lipid transfer proteins. The acceleration of the interlipoprotein transfer caused by prostaglandin E₁ in turn increases the rate of cholesterol esterification in serum. It is suggested that in such a way prostaglandin E₁ may influence the homeostasis of cholesterol.Abbreviations LDL low density lioproteins - HDL high density lipoproteins - PG prostaglandin - ASM anthrylvinyl-labeled sphingomyelin (N-12-(9-anthryl)-11-trans-dodecanoylsphingosin-1-phosphocholine - APC anthrylvinylphosphatidylcholine (1-radyl-2-[(9-anthryl)-11-transdodecanoyl)-sn-glycerophosphocholine - NAP-SM nitroazidophenyl labeled sphingomyelin (N-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sphingosin-1-phosphocholine) - NAP-PC adizophenyl labeled phosphatidylcholine (1-radyl-2-[N-(2-nitro-4azidophenyl)-12-aminododecanoyl]-sn-glycero-3-phosphocholine - DPPC dipalmitoylphosphatidylcholine - P fluorescence polarization - E parameter of tryptophanyl to ASM resonance energy transfer - LEP lipid-exchange protein     

Age-related changes of serum lipoprotein oxidation in rats

Oxidation of low-density lipoprotein (LDL) may be a prelude to atherogenesis and directly age related. To assess whether there may be relationship between age and plasma lipoprotein (LP) oxidation, we studied copper-mediated LP oxidation isolated from the blood of 2 months, 7 months, and 15 months old rats. We determined whether the susceptibility of LP to oxidation might be related to vitamin C levels in serum, vitamin E levels in LP, or the total antioxidant capacity (TAC) of serum or LP. Serum vitamin C content was inversely related to age, malondialdehyde (MDA) propagation rate, and maximum change of MDA concentrations. However, there were no significant relationships between age and serum TAC, LP TAC, serum vitamin E, or the ratio of LP vitamin E to serum vitamin C content. The lag phase of MDA formation was significantly decreased with age and the ratio of LP vitamin E content to serum vitamin C content, increased with age. Maximum change of MDA concentration was positively correlated with the ratio of LP vitamin E contents to serum vitamin C concentration. Thus, as the rat ages, vitamin C status decreases with an increased LP susceptibility to oxidation. It is tempting to speculate that enhanced LP oxidation in older rats may reflect a reduced amount of recycling of LDL vitamin E by serum vitamin C.