Analysis Of High Density Lipoprotein Research Articles

Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study

BackgroundThe previous study investigated whether the functions of small, medium, and large high density lipoprotein (S/M/L-HDL) are correlated with protein changes in mice. Herein, the proteomic and functional analyses of high density lipoprotein (HDL) subclasses were performed in humans and rats.MethodsAfter purifying S/M/L-HDL subclasses from healthy humans (n = 6) and rats (n = 3) using fast protein liquid chromatography (FPLC) with calcium silica hydrate (CSH) resin, the proteomic analysis by mass spectrometry was conducted, as well as the capacities of cholesterol efflux and antioxidation was measured.ResultsOf the 120 and 106 HDL proteins identified, 85 and 68 proteins were significantly changed in concentration among the S/M/L-HDL subclasses in humans and rats, respectively. Interestingly, it was found that the relatively abundant proteins in the small HDL (S-HDL) and large HDL (L-HDL) subclasses did not overlap, both in humans and in rats. Next, by searching for the biological functions of the relatively abundant proteins in the HDL subclasses via Gene Ontology, it was displayed that the relatively abundant proteins involved in lipid metabolism and antioxidation were enriched more in the medium HDL (M-HDL) subclass than in the S/L-HDL subclasses in humans, whereas in rats, the relatively abundant proteins associated with lipid metabolism and anti-oxidation were enriched in M/L-HDL and S/M-HDL, respectively. Finally, it was confirmed that M-HDL and L-HDL had the highest cholesterol efflux capacity among the three HDL subclasses in humans and rats, respectively; moreover, M-HDL exhibited higher antioxidative capacity than S-HDL in both humans and rats.ConclusionsThe S-HDL and L-HDL subclasses are likely to have different proteomic components during HDL maturation, and results from the proteomics-based comparison of the HDL subclasses may explain the associated differences in function.

Identifying causal variants by fine mapping across multiple studies.

Increasingly large Genome-Wide Association Studies (GWAS) have yielded numerous variants associated with many complex traits, motivating the development of "fine mapping" methods to identify which of the associated variants are causal. Additionally, GWAS of the same trait for different populations are increasingly available, raising the possibility of refining fine mapping results further by leveraging different linkage disequilibrium (LD) structures across studies. Here, we introduce multiple study causal variants identification in associated regions (MsCAVIAR), a method that extends the popular CAVIAR fine mapping framework to a multiple study setting using a random effects model. MsCAVIAR only requires summary statistics and LD as input, accounts for uncertainty in association statistics using a multivariate normal model, allows for multiple causal variants at a locus, and explicitly models the possibility of different SNP effect sizes in different populations. We demonstrate the efficacy of MsCAVIAR in both a simulation study and a trans-ethnic, trans-biobank fine mapping analysis of High Density Lipoprotein (HDL).

The biological activity of high-density lipoprotein fractions and their role in the development of cardiovascular diseases

Increasing the human plasma concentration of high-density lipoproteins (HDL) may be part of strategy for control of cardiovascular diseases (CVD). HDL particles vary in their structure, metabolism, and biological activity. The review describes major HDL fractions (subpopulations) and discusses new findings on the antiatherogenic properties of HDL particles. The whole spectrum of HDL fractions, small, dense, protein-rich lipoproteins, has atheroprotective properties that are determined by the presence of specialized groups of proteins and lipids; however, this activity may be decreased in atherogenic lesion. Comprehensive structural and compositional analysis of HDL may provide key information to identify the fractions that have characteristic biological properties and lose their functionality in CVD. These fractions may be also biomarkers for the risk of CVD and hence represent pharmacological targets.

Abstract 17417: ABCC6 Deficiency Promotes Atherosclerosis

Introduction: Vascular calcification is associated with aging and common conditions such as hypercholesterolemia and certain genetic disorders. A molecular pathway inhibiting ectopic calcification is initiated by ABCC6, a membrane transporter primarily expressed in liver. ABCC6 facilitates the cellular efflux of ATP from hepatocytes, which is rapidly converted into pyrophosphate (PPi) in the liver vasculature. ABCC6 is responsible for the majority of the PPi release from the liver, and ~60% of plasma PPi in both in humans and mice. As PPi is a major inhibitor of calcification, mutations in ABCC6 underlie the currently incurable calcification disorder pseudoxanthoma elasticum (PXE), some cases of generalized arterial calcification of infancy (GACI). Hypothesis: Multiple lines of evidence have suggested a pathological synergy between ABCC6 and hyperlipidemia. Methods: We examined crosses between Abcc6-/- and Ldlr-/- mice and serum samples from human PXE patients. Three mouse genotypes were generated Ldlr-/-, Ldlr-/-xAbcc6-/- and Ldlr-/- xAbcc6+/-. Results: When subjected to a high fat diet, mice with reduced or no Abcc6 expression displayed a 2-fold increase of atherosclerotic plaque area (p=0.0001). Interestingly, atherosclerotic plaque was also found in Abcc6-deficient mice fed a normal diet but not in control Ldlr-/- animals. Aortic calcification was not significantly affected by Abcc6 expression except in Ldlr-/-xAbcc6-/- mice fed regular chow (p=0.0001). We observed an increased proliferation of smooth muscle cells of but no increase in monocyte-macrophage infiltration in the plaque of Ldlr-/-xAbcc6-/- mice. Importantly, we determined that the cholesterol efflux from isolated macrophages towards HDL (mediated by ABCG1) was significantly decreased (-28%, p=0.006) in Ldlr-/-xAbcc6-/- mice while the efflux towards ApoA1 (mediated by ABCA1) was unchanged. The analysis of high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions in these mice showed altered profiles similar to those found with human PXE plasma samples. Conclusions: Our results indicated that, in addition to being an important inhibitor of vascular calcification, ABCC6 inhibits atherogenesis by modulating reverse cholesterol transport.

Glycomic Analysis of High Density Lipoprotein Shows a Highly Sialylated Particle

Many of the functional proteins and lipids in high density lipoprotein (HDL) particles are potentially glycosylated, yet very little is known about the glycoconjugates of HDL. In this study, HDL was isolated from plasma by sequential micro-ultracentrifugation, followed by glycoprotein and glycolipid analysis. N-Glycans, glycopeptides, and gangliosides were extracted and purified followed by analysis with nano-HPLC Chip quadrupole time of flight mass spectrometry and MS/MS. HDL particles were found to be highly sialylated. Most of the N-glycans (∼90%) from HDL glycoproteins were sialylated with one or two neuraminic acids (Neu5Ac). The most abundant N-glycan was a biantennary complex type glycan with two sialic acids (Hexose5HexNAc4Neu5Ac2) and was found in multiple glycoproteins using site-specific glycosylation analysis. The observed O-glycans were all sialylated, and most contained a core 1 structure with two Neu5Acs, including those that were associated with apolipoprotein CIII (ApoC-III) and fetuin A. GM3 (monosialoganglioside, NeuAc2–3Gal1–4Glc–Cer) and GD3 (disialoganglioside, NeuAc2–8NeuAc2–3Gal1–4Glc–Cer) were the major gangliosides in HDL. A 60% GM3 and 40% GD3 distribution was observed. Both GM3 and GD3 were composed of heterogeneous ceramide lipid tails, including d18:1/16:0 and d18:1/23:0. This report describes for the first time a glycomic approach for analyzing HDL, highlighting that HDL are highly sialylated particles.

A systems genetic analysis of high density lipoprotein metabolism and network preservation across mouse models

We report a systems genetic analysis of high density lipoprotein (HDL) levels in an F2 intercross between inbred strains CAST/EiJ and C57BL/6J. We previously showed that there are dramatic differences in HDL metabolism in a cross between these strains, and we now report co-expression network analysis of HDL that integrates global expression data from liver and adipose with relevant metabolic traits. Using data from a total of 293 F2 intercross mice, we constructed weighted gene co-expression networks and identified modules (subnetworks) associated with HDL and clinical traits. These were examined for genes implicated in HDL levels based on large human genome-wide associations studies (GWAS) and examined with respect to conservation between tissue and sexes in a total of 9 data sets. We identify genes that are consistently ranked high by association with HDL across the 9 data sets. We focus in particular on two genes, Wfdc2 and Hdac3, that are located in close proximity to HDL QTL peaks where causal testing indicates that they may affect HDL. Our results provide a rich resource for studies of complex metabolic interactions involving HDL. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).

P065 Proteomic Analysis of High-Density Lipoprotein in Chinese Patients with Coronary Heart Disease using Two-dimensional Difference Gel Electrophoresis

P065 Proteomic Analysis of High-Density Lipoprotein in Chinese Patients with Coronary Heart Disease using Two-dimensional Difference Gel Electrophoresis

Abstract 1390: Composition Analysis of High Density Lipoproteins in Patients with extreme HDL-C Levels with or without Ischemic Heart Disease

Objective: We hypothesize that patients with high HDL-C levels and Ischemic Heart Disease (IHD) may have dysfunctional HDL or have unrecognized non-conventional risk factors. Methods: Individuals with verified IHD with either high HDL-C (n=53; female: ≥73.5mg/dL; male: ≥61.9mg/dL) or low HDL-C (n=42; female:≤38.7mg/dL; male: ≤34.1mg/dL) were compared with subjects without IHD and matched HDL-C levels (n=110). All subjects had normal LDL-C (<160mg/dL) and triglyceride levels (<150mg/dL) and none were treated with LDL-C-lowering medications. After removal of apoB lipoproteins, apoA-I, apoA-II, apoE, triglycerides, free cholesterol, cholesteryl ester, and phospholipids were analyzed (Cobas-Fara). HDL and LDL-subclasses were examined by linear gradient gel electrophoresis (Lipoprint®) and by Segmented Gradient Gel Electrophoresis (S-GGE). Cholesterol (RLP-C) and triglyceride (RLP-TG) content of remnant-like lipoprotein particles were measured by an immunoseparation technique. Results: Subjects with high HDL-C levels and IHD had lower apo-AI (p<0.001) and cholesteryl ester (p<0.01) levels compared to controls with high HDL and no IHD. Subjects with low HDL-C and IHD had lower triglyceride (p=0.01) levels compared to controls with matched HDL-C levels and no IHD. The HDL subclass, using S-GGE, with density 1.063–1.100g/ml and diameter 98 –130Å correlated with levels of HDL-C (r 2 =0.66, p<0.0001). LDL subclasses using Lipoprint® exhibited higher fraction of Intermediate Density Lipoproteins (IDL-C) in patients with high HDL-C and IHD when compared with individuals with same HDL-C and no IHD (p=0.006). This was also true in the low HDL-C groups (p=0.01). This was further supported by higher levels of RLP-C and RLP-TG in individuals with high HDL and IHD (p=0.03 and p=0.03, respectively) when compared with respective controls. Conclusions: These results suggest that HDL composition rather than HDL-C levels is important in deciphering the protective role of HDL in IHD and that measurement of remnant-like lipoproteins may be useful for identifying patients with high HDL-C that are still at risk for developing IHD.

Compositional Protein Analysis of High Density Lipoproteins in Hypercholesterolemia by Shotgun LC-MS/MS and Probabilistic Peptide Scoring

A protein of a biological sample is usually quantified by immunological techniques based on antibodies. Mass spectrometry offers alternative approaches that are not dependent on antibody affinity and avidity, protein isoforms, quaternary structures, or steric hindrance of antibody-antigen recognition in case of multiprotein complexes. One approach is the use of stable isotope-labeled internal standards; another is the direct exploitation of mass spectrometric signals recorded by LC-MS/MS analysis of protein digests. Here we assessed the peptide match score summation index based on probabilistic peptide scores calculated by the PHENYX protein identification engine for absolute protein quantification in accordance with the protein abundance index as proposed by Mann and co-workers (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245). Using synthetic protein mixtures, we demonstrated that this approach works well, although proteins can have different response factors. Applied to high density lipoproteins (HDLs), this new approach compared favorably to alternative protein quantitation methods like UV detection of protein peaks separated by capillary electrophoresis or quantitation of protein spots on SDS-PAGE. We compared the protein composition of a well defined HDL density class isolated from plasma of seven hypercholesterolemia subjects having low or high HDL cholesterol with HDL from nine normolipidemia subjects. The quantitative protein patterns distinguished individuals according to the corresponding concentration and distribution of cholesterol from serum lipid measurements of the same samples and revealed that hypercholesterolemia in unrelated individuals is the result of different deficiencies. The presented approach is complementary to HDL lipid analysis; does not rely on complicated sample treatment, e.g. chemical reactions, or antibodies; and can be used for projective clinical studies of larger patient groups.

Reduced HDL particle size as an additional feature of the atherogenic dyslipidemia of abdominal obesity

Reduced plasma HDL cholesterol concentration has been associated with an increased risk of coronary heart disease. However, a low HDL cholesterol concentration is usually not observed as an isolated disorder because this condition is often accompanied by additional metabolic alterations. The objective of this study was to document the relevance of assessing HDL particle size as another feature of the atherogenic dyslipidemia found among subjects with visceral obesity and insulin resistance. For that purpose, an average HDL particle size was computed by calculating an integrated HDL particle size using nondenaturing 4-30% gradient gel electrophoresis. Potential associations between this average HDL particle size versus morphometric and metabolic features of visceral obesity were examined in a sample of 238 men. Results of this study indicated that HDL particle size was a significant correlate of several features of an atherogenic dyslipidemic profile such as increased plasma TG, decreased HDL cholesterol, high apolipoprotein B, elevated cholesterol/HDL cholesterol ratio, and small LDL particles as well as increased levels of visceral adipose tissue (AT) (0.33 < or = absolute value of r < or = 0.61, P < 0.0001). Thus, men with large HDL particles had a more favorable plasma lipoprotein-lipid profile compared with those with smaller HDL particles. Furthermore, men with large HDL particles were also characterized by reduced overall adiposity and lower levels of visceral AT as well as reduced insulinemic-glycemic responses to an oral glucose load. In conclusion, small HDL particle size appears to represent another feature of the high TG- low HDL cholesterol dyslipidemia found in viscerally obese subjects characterized by hyperinsulinemia.

1P038FTIR法による高密度リポタンパク質(HDL)の解析

1P038FTIR法による高密度リポタンパク質(HDL)の解析

Particle size analysis of high density lipoproteins in patients with genetic cholesteryl ester transfer protein deficiency

We investigated the detailed distribution of high-density lipoproteins (HDL) particle size in patients with cholesteryl ester transfer protein (CETP) deficiency. Serum samples pre-stained with Sudan black B were electrophoresed using 4–30% polyacrylamide gradient gels, and the Stokes diameter of HDL particles was determined in 23 patients with genetic CETP deficiency, nine patients with hyperalphalipoproteinemia and seven subjects with normal HDL cholesterol concentrations. The mean Stokes diameter of HDL particles in CETP deficient patients (11.2±0.6 nm) was significantly greater than hyperalphalipoproteinemia (10.7±0.3 nm, P<0.05) and normal subjects (9.5±0.4 nm, P<0.01). A significant relationship was found between mean HDL size and serum CETP mass concentrations ( P<0.05). When the particle size of all detected HDL bands was investigated, extra-large HDL particles larger than 12 nm were found in 14 of the 23 patients with CETP deficiency, which were not found in any of the hyperalphalipoproteinemia patients or normal subjects. Serum low-density lipoproteins (LDL) cholesterol and total cholesterol concentrations were lower in CETP deficiencies with extra-large HDL particles than those in non-carriers ( P<0.01). These results indicate that extra-large HDL may be an index to clarify the relationship between genetic CETP deficiency and atherosclerosis.

Precipitation procedures used to isolate high density lipoprotein with particular reference to effects on apo A-I-only particles and lipoprotein(a)

Analysis of high density lipoprotein (HDL) isolated from serum without major hyper-triglyceridaemia and by five different precipitation methods showed that there were no significant differences in total cholesterol and triglyceride concentrations in the HDL supernatants prepared by the different methods but that free cholesterol, phospholipid, apolipoprotein (apo) A-I and HDL particles containing apo A-I but not apo A-II (LpAI) concentrations were significantly lower for heparin-manganese chloride method 2 (final manganese chloride concentration 92 mmol/1) compared with the other methods. Modest differences in HDL cholesterol, apo A-I and LpAI were observed between heparin-manganese chloride method 1 (final magnesium concentration 46 mmol/1) and the dextran sulphate, phosphotungstate and polyethylene glycol 6000 methods. Lipoprotein(a) (Lp(a)) and apo B were undetectable in the HDL supernatants, indicating that apo B-containing lipoproteins including Lp(a) were essentially completely removed by all the precipitation procedures.

Analysis of high density lipoproteins by a modified gradient gel electrophoresis method.

A high resolution electrophoretic method has been developed to separate plasma high density lipoprotein (HDL) particles by size using 4-30% polyacrylamide agarose (PAA) gradient gels, Sudan black B staining, and laser densitometry. Fourteen distinct HDL bands were observed with HDL-1 being designated as the largest particle and HDL-14 as the smallest particle. HDL-1 was similar in size to ferritin (Stokes diameter 12.2 nm), HDL-8 to catalase (9.2 nm), and HDL-13 to lactate dehydrogenase (8.1 nm). HDL-1 to HDL-7 were found within the density range of HDL2b (d 1.063-1.10 g/ml), HDL-8 to HDL-10 within HDL2a (d 1.10-1.125 g/ml), and HDL-11 to HDL-14 within HDL3 (d 1.125-1.21 g/ml). On immunoblotting, apolipoprotein A-I (apoA-I) was found in all HDL bands examined, being most prominent in HDL-6, 11, and 12. ApoA-II was not detected in HDL bands 1-5, but was present in all other HDL bands and was most prominent in HDL-9. ApoE was detected mainly in HDL bands 1-7, and was observed in only trace amounts in other bands. Lp A-I isolated by immunoaffinity column chromatography from the plasma of five subjects contained five subspecies (HDL-5, 6, and 11-13), while Lp A-I/A-II also had five subspecies (HDL-8, 9, and 11-13) in these subjects. In normal subjects (n = 57) four or five HDL bands were generally observed, with HDL-9, 11, and 12 being the most frequently observed. Mean HDL particle score (method of sizing based on scanning densitometry, where low score indicates large size and high score indicates small size) was significantly correlated (P < 0.001) with the concentrations of HDL cholesterol (r = -0.796), HDL free cholesterol (r = -0.780), HDL cholesteryl ester (r = -0.683), HDL phospholipid (r = -0.663), HDL apoA-I (r = -0.577), and HDL protein (r = -0.459), but not with HDL triglyceride (r = 0.069). In addition, HDL particle score was significantly correlated (P < 0.05) with HDL total mass (r = -0.649), HDL free cholesterol content (% of total mass, r = -0.608), HDL triglyceride content (r = 0.415), HDL phospholipid content (r = -0.359), and HDL protein content (r = 0.295), but not with HDL cholesteryl ester content (r = -0.219) or HDL apoA-I content (r = 0.183).(ABSTRACT TRUNCATED AT 400 WORDS)

NMR analysis of high-density lipoprotein oxidatively modified in vitro

NMR analysis of high-density lipoprotein oxidatively modified in vitro

Heterogeneity of nascent high density lipoproteins secreted by the hepatoma-derived cell line, Hep G2.

Nondenaturing gradient gel analysis of high density lipoproteins (HDL, d 1.063-1.235 g/ml) isolated from Hep G2 24-hr, serum-free conditioned media shows four distinct, reproducible particle subclasses I, II, III, and IV with apparent Stokes' diameters of 13.3, 12.0, 9.5, and 7.4 nm, respectively. Fractions enriched in lipoproteins from each of these subclasses were isolated by either density gradient ultracentrifugation or gel filtration chromatography and characterized. Size and morphology of the isolated subclasses agreed well regardless of isolation procedure. Electron microscopy revealed subclasses I, II, and III to be disc-shaped, and subclass IV to be spherical. The discoidal subclasses were poor in cholesteryl ester and rich in phospholipid and unesterified cholesterol. The larger-sized subclass I particles were enriched in apolipoprotein (apo) E while subclasses II and III had decreasing amounts of apoE and increasing amounts of apoA-I and A-II. The spherical subclass IV particles contained a higher percentage of protein and had a higher ratio of cholesteryl ester to unesterified cholesterol than that found in the other subclasses. Subclass IV contained predominantly apoA-I. The subclasses isolated from Hep G2 HDL appear to share many similarities with those isolated from patients with lecithin:cholesterol acyltransferase deficiency and are therefore potentially useful in examining the transformation of nascent HDL particles to mature circulating plasma forms.

Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis.

In order to determine the effects of a plasma phospholipid transfer protein on the transfer of phospholipids from very low density lipoproteins (VLDL) to high density lipoproteins (HDL) during lipolysis, biosynthetically labeled rat 32P-labeled VLDL was incubated with human HDL3 and bovine milk lipoprotein lipase (LPL) in the presence of the plasma d greater than 1.21 g/ml fraction or a partially purified human plasma phospholipid transfer protein (PTP). The addition of either the PTP or the d greater than 1.21 g/ml fraction resulted in a 2- to 3-fold stimulation of the transfer of phospholipid radioactivity from VLDL into HDL during lipolysis. In the absence of LPL, the PTP caused a less marked stimulation of transfer of phospholipid radioactivity. Both the d greater than 1.21 g/ml fraction and the PTP enhanced the transfer of VLDL phospholipid mass into HDL, but the percentage transfer of phospholipid radioactivity was greater than that of phospholipid mass, suggesting stimulation of both transfer and exchange processes. Stimulation of phospholipid exchange was confirmed in experiments where PTP was found to augment transfer of [14C]phosphatidylcholine radioactivity from HDL to VLDL during lipolysis. In experiments performed with human VLDL and human HDL3, both the d greater than 1.21 g/ml fraction and the PTP were found to stimulate phospholipid mass transfer from VLDL into HDL during lipolysis. Analysis of HDL by non-denaturing polyacrylamide gradient gel electrophoresis showed that enhanced lipid transfer was associated with only a slight increase in particle size, suggesting incorporation of lipid by formation of new HDL particles. In conclusion, the plasma d greater than 1.21 g/ml fraction and a plasma PTP enhance the net transfer of VLDL phospholipids into HDL and also exchange of the phospholipids of VLDL and HDL. Both the transfer and exchange activities of PTP are stimulated by lipolysis.

Protein content and composition of human high density lipoprotein subfractions

High density lipoprotein (HDL) was fractionated by ion exchange column chromatography using a continuous NaCl gradient of 0.06 – 0.13 M. It was found that, on the basic C apoprotein content, HDL is comprised of 3 subfractions. All 3 subfractions contain apo A-I and apo A-II but the apo A-I/apo A-II ratio is different in each subfraction and in the case of subfraction c, that fraction which eluted at highest NaCl concentrations, the A-I/A-II ratio varied even within this subfraction. Subfraction a contained no C apoprotein, subfraction b contained apo C-II and apo C-III-1 but no apo C-III-2 while subfraction c contained apo C-III-2 and trace amounts of apo C-II and C-III-1. Analysis of HDL 2 and HDL 3 shows that both contain all 3 lipoprotein subfractions, but in differing quantities.