Direct Estimation of HDL-Mediated Cholesterol Efflux Capacity from Serum

Abstract BACKGROUND HDL-mediated cholesterol efflux capacity (HDL-CEC) is a functional attribute that may have a protective role in atherogenesis. However, the estimation of HDL-CEC is based on in vitro cell assays that are laborious and hamper large-scale phenotyping. METHODS Here, we present a cost-effective high-throughput nuclear magnetic resonance (NMR) spectroscopy method to estimate HDL-CEC directly from serum. We applied the new method in a population-based study of 7603 individuals including 574 who developed incident coronary heart disease (CHD) during 15 years of follow-up, making this the largest quantitative study for HDL-CEC. RESULTS As estimated by NMR-spectroscopy, a 1-SD higher HDL-CEC was associated with a lower risk of incident CHD (hazards ratio, 0.86; 95%CI, 0.79–0.93, adjusted for traditional risk factors and HDL-C). These findings are consistent with published associations based on in vitro cell assays. CONCLUSIONS These corroborative large-scale findings provide further support for a potential protective role of HDL-CEC in CHD and substantiate this new method and its future applications.

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Direct estimation of HDL-mediated cholesterol efflux capacity from serum

10.1101/396929 ◽

2018 ◽

Author(s):

Sanna Kuusisto ◽

Michael V. Holmes ◽

Pauli Ohukainen ◽

Antti J. Kangas ◽

Mari Karsikas ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Cholesterol Efflux ◽

Large Scale ◽

Density Lipoprotein ◽

Population Based ◽

Protective Role ◽

New Method ◽

Cholesterol Efflux Capacity ◽

Cell Assays

AbstractHigh-density lipoprotein mediated cholesterol efflux capacity (HDL-CEC) is a functional attribute that may have a protective role in atherogenesis. However, the estimation of HDL-CEC is based on in vitro cell assays that are laborious and hamper large-scale phenotyping. Here, we present a cost-effective high-throughput nuclear magnetic resonance (NMR) spectroscopy method to estimate HDL-CEC directly from serum. We applied the new method in a population-based study of 7,603 individuals including 574 who developed incident coronary heart disease (CHD) during 15 years of follow-up, making this the largest quantitative study for HDL-CEC. As estimated by NMR-spectroscopy, a 1-SD higher HDL-CEC was associated with a lower risk of incident CHD (hazards ratio 0.86; 95%CI 0.79-0.93, adjusted for traditional risk factors and HDL-C). These findings are consistent with published associations based on in vitro cell assays. These corroborative large-scale findings provide further support for a potential protective role of HDL-CEC in CHD, and substantiate this new method and its future applications.

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Abstract 17232: Comparison of Methods for the Measurement of Cholesterol Efflux Capacity of Plasma HDL With J774 Mouse Macrophages Reveals Superiority of the Radioactive Cholesterol Method over the BODIPY-Cholesterol-2 Method

Circulation ◽

10.1161/circ.132.suppl_3.17232 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

David Rhainds ◽

Renaud Julien ◽

Boulé Marie ◽

Denis Maxime ◽

Rhéaume Eric ◽

...

Keyword(s):

Cholesterol Efflux ◽

Hdl Cholesterol ◽

Fold Increase ◽

Strongly Correlated ◽

Response Curves ◽

Complex Samples ◽

Cholesterol Efflux Capacity ◽

Mouse Macrophages ◽

Efflux Time

Recent clinical studies suggest that raising HDL-cholesterol (HDL-C) concentration is insufficient to lower cardiovascular risk and that protection derives from other characteristics of HDL particles. One such characteristic that can be measured in vitro is the cholesterol efflux capacity of plasma, an assay where HDL accepts labelled cholesterol from macrophages. Recently, a method for the measurement of cholesterol efflux with a fluorescent tracer, BODIPY-cholesterol-2 (BC2), has been proposed as an alternative to the radioactive method. We undertook a systematic comparison of BC2 and 3H-cholesterol methods with J774 macrophages in basal and cAMP-stimulated conditions with purified acceptors, plasma from healthy volunteers and patients with myocardial infarction of the Montreal Heart Institute Biobank. Dose-response curves show higher affinity of BC2 vs. 3H cholesterol with apoA-I, HDL and apoB-depleted plasma (all p<0.01) and a higher maximal efflux for HDL and depleted plasma (both p<0.001) in stimulated conditions. This was reflected in a faster kinetics of BC2 efflux compared to 3H-cholesterol efflux (time for half-maximal efflux 4.1h vs. 10.2 h) and resulted in a 2.5-fold increase of BC2 maximal efflux (p<0.01). With 50 normolipidemic plasmas (2.8%, 4h efflux), the two methods were not correlated in basal conditions (r2=0.079) and strongly correlated in stimulated conditions (r2=0.830). BC2 values did not correlate with HDL-C in all conditions, contrary to 3H cholesterol values (p<0.01). Using more complex samples from patients with MI (n=115), the correlations were modest in basal (r2=0.219) and stimulated (r2=0.400) conditions. Differences between controls and MI cases showed reduced significance with BC-2 compared to 3H cholesterol. Lastly, in macrophages examined under the confocal microscope, BC2 labels vesicular structures colocalized with filipin, a free cholesterol marker, suggesting endosomal loading of BC2. Thus, the BC2 method is not equivalent to the classic 3H cholesterol method for cholesterol efflux measurement.

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Abstract 319: Dalcetrapib-Mediated Modulation of Cholesteryl Ester Transfer Protein Activity Increases HDL-Cholesterol, Apolipoprotein A-I Levels and Cholesterol Efflux Capacity in Chow-Fed Rabbits

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a319 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Mathieu R Brodeur ◽

David Rhainds ◽

Daniel Charpentier ◽

Téodora Mihalache-Avram ◽

Cyrille Maugeais ◽

...

Keyword(s):

Cholesteryl Ester ◽

Cholesteryl Ester Transfer Protein ◽

Cholesterol Efflux ◽

Hdl Cholesterol ◽

Lipid Transfer ◽

Protein Activity ◽

Cholesterol Efflux Capacity ◽

Transfer Protein

Introduction: A potential approach to reduce CV risk is to increase HDL-C levels. This could be achieved by reducing cholesteryl ester transfer protein (CETP) activity. Dalcetrapib, which modulates CETP activity by changing its conformation and raises HDL-C without inhibiting CETP-induced pre-β-HDL formation in humans, was shown to decrease progression of atherosclerosis in rabbits. Hypothesis: Investigate the modifications of HDL particle size distribution and cholesterol efflux capacity of serum produced by dalcetrapib in normocholesterolemic rabbits. Methods: New Zealand white rabbits were treated with dalcetrapib (300 mg/kg as food admix) or placebo for 14 days. We evaluated CETP conformation and mass by ELISAs (including antibodies sensitive to conformational change), CETP activity by fluorescent lipid transfer, lipid profile and apoA-I distribution in HDL subclasses by 2D-non denaturing gradient gels (2D-NDGGE). Cholesterol efflux capacity of rabbit sera was determined after loading cells with 3 H-free cholesterol, using HepG2 hepatocytes to measure SR-BI-dependent efflux and by inducing ABCA1 or ABCG1 expression in BHK cells. Results: Dalcetrapib modified the conformation of rabbit CETP in vitro and in vivo and, after 14 days, this was associated with increased CETP mass (+50%, p<0.001) and reduced CETP activity (-86%, p<0.001). Total cholesterol was increased with dalcetrapib (+178%, p<0.001), due to a higher HDL-C level. In contrast, dalcetrapib reduced LDL-C and triglycerides by 41% (p<0.01) and 48% (p<0.001). Serum analysis by 2D-NDGGE showed that total rabbit apoA-I was increased 1.7- fold in animals treated with dalcetrapib. This was associated with an increase in large HDL but also in small α-migrating HDL with pre-β-HDL size. Cholesterol efflux assays showed that ABCA1-, ABCG1- and SR-BI-dependent efflux were all increased in dalcetrapib-treated rabbits (+24%, p=0.038; +21%, p=0.021; +44%, p<0.001). Conclusion: Modulation of CETP activity and conformation by dalcetrapib increases HDL-C and apoA-I levels and affects apoA-I distribution in HDL subclasses. These changes are associated with increased cholesterol efflux capacity, suggesting that HDL functionality is preserved in dalcetrapib-treated chow-fed rabbits.

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Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity

European Journal of Clinical Investigation ◽

10.1111/j.1365-2362.2006.01673.x ◽

2006 ◽

Vol 36 (8) ◽

pp. 552-559 ◽

Cited By ~ 20

Author(s):

S. Mweva ◽

J. L. Paul ◽

M. Cambillau ◽

D. Goudouneche ◽

P. Beaune ◽

...

Keyword(s):

Human Serum ◽

Serum Cholesterol ◽

Cholesterol Efflux ◽

Cholesterol Efflux Capacity ◽

Cellular Models

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Proof of concept for quantitative urine NMR metabolomics pipeline for large-scale epidemiology and genetics

International Journal of Epidemiology ◽

10.1093/ije/dyy287 ◽

2019 ◽

Vol 48 (3) ◽

pp. 978-993 ◽

Cited By ~ 6

Author(s):

Tuulia Tynkkynen ◽

Qin Wang ◽

Jussi Ekholm ◽

Olga Anufrieva ◽

Pauli Ohukainen ◽

...

Keyword(s):

Nmr Spectroscopy ◽

Large Scale ◽

Molecular Data ◽

Population Based ◽

Urine Samples ◽

Metabolomics Data ◽

Urine Metabolites ◽

Nmr Metabolomics ◽

Genome Wide ◽

Metabolic Information

Abstract Background Quantitative molecular data from urine are rare in epidemiology and genetics. NMR spectroscopy could provide these data in high throughput, and it has already been applied in epidemiological settings to analyse urine samples. However, quantitative protocols for large-scale applications are not available. Methods We describe in detail how to prepare urine samples and perform NMR experiments to obtain quantitative metabolic information. Semi-automated quantitative line shape fitting analyses were set up for 43 metabolites and applied to data from various analytical test samples and from 1004 individuals from a population-based epidemiological cohort. Novel analyses on how urine metabolites associate with quantitative serum NMR metabolomics data (61 metabolic measures; n = 995) were performed. In addition, confirmatory genome-wide analyses of urine metabolites were conducted (n = 578). The fully automated quantitative regression-based spectral analysis is demonstrated for creatinine and glucose (n = 4548). Results Intra-assay metabolite variations were mostly <5%, indicating high robustness and accuracy of urine NMR spectroscopy methodology per se. Intra-individual metabolite variations were large, ranging from 6% to 194%. However, population-based inter-individual metabolite variations were even larger (from 14% to 1655%), providing a sound base for epidemiological applications. Metabolic associations between urine and serum were found to be clearly weaker than those within serum and within urine, indicating that urinary metabolomics data provide independent metabolic information. Two previous genome-wide hits for formate and 2-hydroxyisobutyrate were replicated at genome-wide significance. Conclusion Quantitative urine metabolomics data suggest broad novelty for systems epidemiology. A roadmap for an open access methodology is provided.

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In Vivo Inflammation Does Not Impair ABCA1-Mediated Cholesterol Efflux Capacity of HDL

Cholesterol ◽

10.1155/2012/610741 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Remco Franssen ◽

Alinda W. M. Schimmel ◽

Sander I. van Leuven ◽

Simone C. S. Wolfkamp ◽

Erik S. G. Stroes ◽

...

Keyword(s):

Severe Sepsis ◽

Inflammatory Disease ◽

Cholesterol Efflux ◽

Chronic Inflammatory Disease ◽

Cholesterol Efflux Capacity ◽

Hdl Function ◽

In Vivo Inflammation ◽

The Impact

HDL provides atheroprotection by facilitating cholesterol efflex from lipid-laden macrophages in the vessel wall. In vitro studies have suggested impaired efflux capacity of HDL following inflammatory changes. We assessed the impact of acute severe sepsis and mild chronic inflammatory disease on the efflux capacity of HDL. We hypothesize that a more severe inflammatory state leads to stronger impaired cholesterol efflux capacity. Using lipid-laden THP1 cells and fibroblasts we were able to show that efflux capacity of HDL from both patients with severe sepsis or with Crohn's disease (active or in remission), either isolated using density gradient ultracentrifugation or using apoB precipitation, was not impaired. Yet plasma levels of HDL cholesterol and apoA-I were markedly lower in patients with sepsis. Based on the current observations we conclude that inflammatory disease does not interfere with the capacity of HDL to mediate cholesterol efflux. Our findings do not lend support to the biological relevance of HDL function changes in vitro.

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Molecular Patterns of Extreme and Persistent Cholesterol Efflux Capacity

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.315648 ◽

2021 ◽

Vol 41 (10) ◽

pp. 2588-2597

Author(s):

Ayea El-Ghazali ◽

Sneha Deodhar ◽

Suzanne Saldanha ◽

Brooke Smyth ◽

Mark Izbrand ◽

...

Keyword(s):

Cardiovascular Disease ◽

Size Distribution ◽

Cholesterol Efflux ◽

Total Phospholipid ◽

Density Lipoprotein ◽

Population Based ◽

Size Exclusion ◽

Atherosclerotic Cardiovascular Disease ◽

Cholesterol Efflux Capacity ◽

Cardiovascular Biomarker

Objective: Cholesterol efflux capacity (CEC), the ability of extracellular acceptors to pick-up cholesterol from macrophages, is a clinically relevant cardiovascular biomarker. CEC is inversely associated with incident atherosclerotic cardiovascular disease events. However, CEC is only modestly associated with HDL-C (high-density lipoprotein cholesterol) levels, which may explain the failure of HDL-C raising therapies to improve atherosclerotic cardiovascular disease outcomes. Determinants of variation in CEC are not well understood. Thus, we sought to establish whether extreme high and low CEC is a robust persistent phenotype and to characterize associations with cholesterol, protein, and phospholipids across the particle size distribution. Approach and Results: CEC was previously measured in 2924 participants enrolled in the Dallas Heart Study, a multi-ethnic population-based study from 2000 to 2002. We prospectively recruited those who were below the 10th and above 90th percentile of CEC. Our study revealed that extreme low and high CEC are persistent, robust phenotypes after 15 years of follow-up. Using size exclusion chromatography, CEC to fractionated plasma depleted of apolipoprotein B (fraction-specific CEC) demonstrated significant differences in CEC patterns between persistent high and low efflux groups. Fraction-specific CEC was correlated with fraction-specific total phospholipid but not apolipoprotein A-I, cholesterol, or total protein. These correlations varied across the size distribution and differed among persistent high versus low efflux groups. Conclusions: Extreme high and low CEC are persistent and robust phenotypes. CEC patterns in fractionated plasma reveal marked variation across the size distribution. Future studies are warranted to determine specific molecular species linked to CEC in a size-specific manner.

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Relationship between HDL Cholesterol Efflux Capacity, Calcium Coronary Artery Content, and Antibodies against ApolipoproteinA-1 in Obese and Healthy Subjects

Journal of Clinical Medicine ◽

10.3390/jcm8081225 ◽

2019 ◽

Vol 8 (8) ◽

pp. 1225 ◽

Cited By ~ 6

Author(s):

Nicolas Vuilleumier ◽

Sabrina Pagano ◽

Fabrizio Montecucco ◽

Alessandra Quercioli ◽

Thomas H. Schindler ◽

...

Keyword(s):

Coronary Artery ◽

Cholesterol Efflux ◽

Positive Association ◽

Hdl Cholesterol ◽

Cholesterol Content ◽

Cholesterol Efflux Capacity ◽

Clinical Observations ◽

Framingham Risk ◽

Obese Subjects

Aims: To explore the associations between cholesterol efflux capacity (CEC), coronary artery calcium (CAC) score, Framingham risk score (FRS), and antibodies against apolipoproteinA-1 (anti-apoA-1 IgG) in healthy and obese subjects (OS). Methods and Results: ABCA1-, ABCG1-, passive diffusion (PD)-CEC and anti-apoA-1 IgG were measured in sera from 34 controls and 35 OS who underwent CAC score determination by chest computed tomography. Anti-apoA-1 IgG ability to modulate CEC and macrophage cholesterol content (MCC) was tested in vitro. Controls and OS displayed similar ABCG1-, ABCA1-, PD-CEC, CAC and FRS scores. Logistic regression analyses indicated that FRS was the only significant predictor of CAC lesion. Overall, anti-apoA-1 IgG were significantly correlated with ABCA1-CEC (r = 0.48, p < 0.0001), PD-CEC (r = −0.33, p = 0.004), and the CAC score (r = 0.37, p = 0.03). ABCA1-CEC was correlated with CAC score (r = 0.47, p = 0.004) and FRS (r = 0.18, p = 0.29), while PD-CEC was inversely associated with the same parameters (CAC: r = −0.46, p = 0.006; FRS: score r = −0.40, p = 0.01). None of these associations was replicated in healthy controls or after excluding anti-apoA-1 IgG seropositive subjects. In vitro, anti-apoA-1 IgG inhibited PD-CEC (p < 0.0001), increased ABCA1-CEC (p < 0.0001), and increased MCC (p < 0.0001). Conclusions: We report a paradoxical positive association between ABCA1-CEC and the CAC score, with the latter being inversely associated with PD in OS. Corroborating our clinical observations, anti-apoA-1 IgG enhanced ABCA1 while repressing PD-CEC, leading to MCC increase in vitro. These results indicate that anti-apoA-1 IgG have the potential to interfere with CEC and macrophage lipid metabolism, and may underpin paradoxical associations between ABCA1-CEC and cardiovascular risk.

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Effective Islet Isolation Method with Extremely High Islet Yields from Adult Pigs

Cell Transplantation ◽

10.3727/000000005783982512 ◽

2005 ◽

Vol 14 (10) ◽

pp. 757-762 ◽

Cited By ~ 36

Author(s):

Yukihide Yonekawa ◽

Shinichi Matsumoto ◽

Teru Okitsu ◽

Takashi Arata ◽

Yasuhiro Iwanaga ◽

...

Keyword(s):

Large Scale ◽

New Method ◽

Human Pancreas ◽

Islet Isolation ◽

Isolation Method ◽

Porcine Pancreas ◽

Porcine Islet ◽

Single Donor

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase™ HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 ± 213,486 IE) (mean ± SE) before purification and 800,000 IE (879,815 ± 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 ± 11,532 IE, p < 0.01) and the Ricordi method (317,073 ± 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.

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Prevention of Toxic Effects of Mycotoxins by Means of Nonnutritive Adsorbent Compounds

Journal of Food Protection ◽

10.4315/0362-028x-59.6.631 ◽

1996 ◽

Vol 59 (6) ◽

pp. 631-641 ◽

Cited By ~ 131

Author(s):

ANTONIO-JAVIER RAMOS ◽

JOHANA FINK-GREMMELS ◽

ENRIQUE HERNÁNDEZ

Keyword(s):

Large Scale ◽

Cost Effective ◽

Toxic Effects ◽

Animal Products ◽

Recent Approach ◽

Calcium Aluminosilicate ◽

Exchange Resins ◽

Biological Methods

Mycotoxins comprise a family of fungal toxins, many of which have been implicated as chemical progenitors of toxicity in man and animals. The most thoroughly studied are the aflatoxins. A variety of physical, chemical, and biological methods to counteract the mycotoxin problem have been reported, but large-scale, practical, and cost-effective methods for detoxifying mycotoxin-containing feedstuffs are currently not available. The most recent approach to the problem has been the addition to the animal's diet of nonnutritive sorbents that sequester mycotoxins and reduce their gastrointestinal absorption, avoiding their toxic effects on livestock and toxin carryover into animal products. This review comments on the in vitro efficacy of several of the adsorbents assayed, and their in vivo applications in a range of animals will be discussed. The sorbents reviewed are activated charcoal, bentonite, zeolite, hydrated sodium calcium aluminosilicate (HSCAS) and a wide variety of clays and synthetic ion-exchange resins.

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