Preparative Ultracentrifugation Research Articles

Comparison of low-density lipoprotein cholesterol equations in patients with dyslipidaemia receiving cholesterol ester transfer protein inhibition

Abstract Background Low-density lipoprotein (LDL-C) lowering is imperative in cardiovascular disease prevention. Effectively translating the evidence for LDL-C lowering to maximize clinical and public health benefits depends on the availability of accurate LDL-C results from clinical laboratories to guide therapy. Furthermore, prior work has raised the possibility that cholesterol esterase transfer protein (CETP) inhibition could interfere with accurate assessment of LDL-C. Purpose We aimed to compare accuracy of three clinically implemented LDL-C equations in a clinical trial of CETP inhibition. Methods Men and women aged 18–75 years with dyslipidaemia were recruited from 17 sites in the Netherlands and Denmark. Patients were randomly assigned to one of nine groups using various combinations of the CETP inhibitor TA-8995, statin therapy, and placebo. In pooled measurements over 12 weeks, we calculated LDL-C by the Friedewald, Martin/Hopkins, and Sampson equations, and compared values with preparative ultracentrifugation (PUC) LDL-C as the reference measure (also known as “beta quantification”). Based on prior literature and dyslipidaemia guidelines, we examined correct classifications across the LDL-C 1.81 mmol/L cutpoint in the subgroup of patients with triglycerides of 1.69–4.51 mmol/L. Results The analysis included 242 patients contributing 921 observations. The full distributions of differences between LDL-C estimates and PUC are shown in Figure 1. Overall median LDL-C differences between estimates and PUC were small: Friedewald, 0.00 (25th, 75th: −0.10, 0.08) mmol/L; Martin/Hopkins, 0.02 (−0.08, 0.10) mmol/L; and Sampson, 0.05 (−0.03, 0.15) mmol/L. In the subgroup with estimated LDL-C <1.81 mmol/L and triglycerides 1.69–4.51 mmol/L, the Friedewald equation underestimated LDL-C with a median difference versus PUC of −0.25 (−0.33, −0.10) mmol/L, whereas the Martin/Hopkins equation corrected this issue with a median difference of 0.00 (−0.08, 0.10) mmol/L and the Sampson equation showed tendency towards underestimation with a median difference of −0.06 (−0.13, 0.00) mmol/L. In patients with triglyceride levels of 1.69–4.51 mmol/L, Figure 2 shows the proportion of LDL-C levels classified by the equations as < or ≥1.81 mmol/L that were correctly classified when compared with PUC. All three equations showed >95% accuracy when estimated LDL-C levels were ≥1.81 mmol/L. However, the proportion of LDL-C observations <1.81 mmol/L that were correctly classified compared with PUC was 71.4% by Friedewald versus 100.0% by Martin/Hopkins and 93.1% by Sampson. Conclusion In Europeans with dyslipidaemia, our analysis shows improved accuracy in LDL-C when using contemporary LDL-C equations over the Friedewald equation, particularly when using the Martin/Hopkins equation. High levels of accuracy with the Martin/Hopkins equation were seen in individuals treated with CETP inhibition and even in the context of low LDL-C and high triglyceride levels. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Dezima and NewAmsterdam Pharma

Obicetrapib Lowers LDL-C in Patients Taking High Intensity Statins - Results from the ROSE Clinical Trial

Lead Author's Financial Disclosures New Amsterdam Pharma. Study Funding NewAmsterdam Pharma. Background/Synopsis Mendelian randomization studies have predicted that cholesteryl ester transfer protein (CETP) inhibition would reduce the risk of cardiovascular events. This was confirmed in REVEAL; by lowering LDL-C, anacetrapib was associated with a reduction in cardiovascular events. Early studies of the selective CETP inhibitor obicetrapib demonstrated LDL-C lowering up to 45% as monotherapy and 68% in combination with moderate intensity statin therapy. Objective/Purpose To assess the effects of obicetrapib on LDL-C and lipid parameters in patients treated with high intensity statins. Methods Patients (n=120) with an LDL-C ≥70 mg/dL, despite treatment with high intensity statins, were randomized to treatment with obicetrapib 5 or 10 mg or matching placebo for 8 weeks. The primary endpoint was the difference between groups in percent change in LDL-C from baseline to week 8. Secondary endpoints included changes in ApoB, non-HDL-C and HDL-C. Exploratory analyses evaluated differences in Friedewald and preparative ultracentrifugation measurement (PUC) of LDL-C. Safety assessment included biochemistry, vital signs, physical examinations, and adverse events. Results Patients had a median LDL-C of 88 mg/dL at baseline. Compared with placebo, obicetrapib produced greater dose-dependent lowering of LDL-C (up to 50.8%, P<0.0001), which was comparable between PUC and Friedewald measures. Obicetrapib also produced greater lowering of apoB (up to 29.8%, P<0.0001), non-HDL-C (up to 44.4%, P<0.0001), Lp(a) (up to 56.5%, P<0.0001) and raising of HDL-C (up to 165%, P<0.0001) in a dose-dependent fashion compared with placebo. Fewer patients in the obicetrapib arms (26%) experienced treatment-emergent adverse events than in the placebo arm (48%). No patients in either obicetrapib arm experienced a treatment-emergent SAE. There were no clinically significant changes in laboratory parameters, vital signs, or physical examinations in any study group. Conclusions Obicetrapib at doses of 5 and 10 mg produced robust decreases in LDL-C, ApoB, Lp(a) and Non-HDL-C and increases in HDL-C compared to placebo in patients treated with high intensity statins and was safe and well tolerated. New Amsterdam Pharma.

Obicetrapib Lowers LDL-C in Patients Taking High Intensity Statins - Results from the ROSE Clinical Trial

<h3>Lead Author's Financial Disclosures</h3> Nothing to disclose. <h3>Study Funding</h3> NewAmsterdam Pharma. <h3>Background/Synopsis</h3> Mendelian randomization studies have predicted that cholesteryl ester transfer protein (CETP) inhibition would reduce the risk of cardiovascular events. This was confirmed in REVEAL; by lowering LDL-C, anacetrapib was associated with a reduction in cardiovascular events. Early studies of the selective CETP inhibitor obicetrapib demonstrated LDL-C lowering up to 45% as monotherapy and 68% in combination with moderate intensity statin therapy. <h3>Objective/Purpose</h3> To assess the effects of obicetrapib on LDL-C and other lipid parameters in patients treated with high intensity statins. <h3>Methods</h3> Patients (n=120) with an LDL-C ≥70 mg/dL, despite treatment with high intensity statins, were randomized to treatment with obicetrapib 5 or 10mg or matching placebo for 8 weeks. The primary endpoint was the difference between groups in percent change in LDL-C from baseline to week 8. Secondary endpoints included changes in ApoB, non-HDL-C and HDL-C. Exploratory analyses evaluated differences in Friedewald and preparative ultracentrifugation measurement (PUC) of LDL-C. Safety assessment included biochemistry, vital signs, physical examinations, and adverse events. <h3>Results</h3> Patients had a median LDL-C of 88mg/dL at baseline. Compared with placebo, obicetrapib produced greater dose-dependent lowering of LDL-C (up to 50.8%, P<0.0001), which was comparable between PUC and Friedewald measures. Obicetrapib also produced greater lowering of apoB (up to 29.8%, P<0.0001) and non-HDL-C (up to 44.4%, P<0.0001) and raising of HDL-C (up to 165%, P<0.0001) in a dose-dependent fashion compared with placebo. (Table) Fewer patients in the obicetrapib arms (26%) experienced treatment emergent adverse events than in the placebo arm (48%). No patients in either obicetrapib arm experienced a treatment emergent SAE. There were no clinically significant changes in laboratory parameters, vital signs, or physical examinations in any study group. <h3>Conclusions</h3> Obicetrapib at doses of 5 and 10mg produced robust decreases in LDL-C, ApoB and Non-HDL-C and increases in HDL-C compared to placebo in patients treated with high intensity statins and was safe and well tolerated.

Effect of PCSK9 inhibition with evolocumab on lipoprotein subfractions in familial dysbetalipoproteinemia (type III hyperlipidemia).

Familial dysbetalipoproteinemia (FDBL) is a rare inborn lipid disorder characterized by the formation of abnormal triglyceride- and cholesterol-rich lipoproteins (remnant particles). Patients with FDBL have a high risk for atherosclerotic disease. The effect of PCSK9 inhibition on lipoproteins and its subfractions has not been evaluated in FDBL. Three patients (65±7 years, 23±3 kg/m2, 2 females) with FDBL (diagnosed by isoelectrofocusing) and atherosclerosis (coronary and/or cerebro-vascular and/or peripheral arterial disease) resistant or intolerant to statin and fibrate therapy received evolocumab (140mg every 14 days). In addition to a fasting lipid profile (preparative ultracentrifugation), apoB and cholesterol concentrations were determined in 15 lipoprotein-subfractions (density gradient ultracentrifugation; d 1.006-1.21g/ml) before and after 12 weeks of evolocumab treatment. Patients with LDL-hypercholesterolemia (n = 8, 56±8 years, 31±7 kg/m2) and mixed hyperlipidemia (n = 5, 68±12 years, 30±1 kg/m2) also receiving evolocumab for 12 weeks were used for comparison. All patients tolerated PCSK9 inhibition well. PCSK9 inhibitors reduced cholesterol (29-37%), non-HDL-cholesterol (36-50%) and apoB (40-52%) in all patient groups including FDBL. In FDBL, PCSK9 inhibition reduced VLDL-cholesterol and the concentration of apoB containing lipoproteins throughout the whole density spectrum (VLDL, IDL, remnants, LDL). Lipoprotein(a) was decreased in all patient groups to a similar extent. This indicates that the dominant fraction of apoB-containing lipoproteins is reduced with PCSK9 inhibition, i.e. LDL in hypercholesterolemia and mixed hyperlipidemia, and cholesterol-rich VLDL, remnants and LDL in FDBL. PCSK9 inhibition may be a treatment option in patients with FDBL resistant or intolerant to statin and/or fibrate therapy.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer.

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.

Features and quantitative characteristics of the interaction of benzo[a]pyrene with fractions of the human blood plasma lipoproteins

The paper considers the features and quantitative characteristics of the interaction of human blood plasma low (LDL), very low (VLDL) and high density lipoproteins (HDL) with a polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P). Material and methods. The studies were performed using preparative ultracentrifugation of human plasma lipoprotein fractions and fluorescence spectroscopy. Results. The spectral characteristics of tryptophan fluorescence of VLDL, LDL and HDL fractions during titration with B[a]P solution are presented. The interaction of LP fractions with B[a]P was accompanied by quenching of tryptophanil fluorescence: the greatest decrease in fluorescence at saturating concentrations of B[a]P was noted for the HDL fraction — over 80 %; the effect of B[a]P was weaker for LDL (65-74 %) and for VLDL (50-60 %) of the initial level. Based on the results of the tryptophan fluorescence quenching curves and taking into account the molecular weights, quantitative characteristics of the interaction of the LP fractions with B[a]P were obtained: the association constants were of the order of (1.3 - 2.5) × 10-6 M, and the number of binding sites for B[a] P on LP particles ranged from 19 for HDL and up to 43 for VLDL. Conclusion. The quantitative characteristics of the interaction obtained for the complexes of LP with B[a]P allow us to consider the LP of blood plasma as real transport forms for B[a]P and other hydrophobic compounds into the cells of organs and tissues of the body.

Assembly Control at a Low Péclet Number in Ultracentrifugation for Uniformly Sized Nanoparticles

The intrinsic high diffusion rate of colloids at low Péclet number results in an extremely fast crystallization process and instant formation of colloidal crystals, even at an ultracentrifugal field of extremely high intensity. By introducing a small number of clusters in sedimention, it should be possible to slow down the crystallization process, thus making the assembly order tunable in preparative ultracentrifugation experiments. Here, we used sodium dodecyl sulfate-stabilized polystyrene nanoparticles (with a size dispersity of 1.07) dispersed in a solution of high ionic strength. Sedimentation and assembly of these nanoparticles were done using preparative ultracentrifugation at various angular velocities. The sedimentation process was also analyzed in situ by analytical ultracentrifugation in real time. By creating as low as 3% of clusters into these nearly uniformly sized polystyrene nanoparticle dispersions during the sedimentation process, the superstructure order becomes easily tunable between glassy and crystalline. Theoretical calculations complemented the experiments to explain the mechanism of cluster formation in sedimentation. This work provides a novel methodology to produce superstructures with a tunable packing order for colloids at low Péclet number.

Perspectives on the Changing Landscape of Measuring Cardiovascular Risk Related to LDL.

The clinical laboratory plays a critical role in the assessment of atherosclerotic cardiovascular disease (ASCVD)9 risk. Since the 1970s, this laboratory assessment has been primarily through tests in the lipid panel, which included cholesterol, triglycerides (TG), and HDL cholesterol (HDL-C) based on fasting serum or plasma specimens. Until the advent of direct LDL cholesterol (LDL-C) tests approximately 20 years ago, LDL-C was almost exclusively estimated by the Friedewald equation (valid when TG <400 mg/dL or <4.5 mmol/L), and to date the vast majority of LDL-C is still calculated. This testing paradigm has undergone substantial changes over the past decade, with the emergence of new research and guidelines recommending alternate approaches for the laboratory assessment of ASCVD risk. As early as 2009, various clinical laboratory associations started recommending the use of a nonfasting specimen for routine lipid panels as a result of several studies supporting its use in cardiovascular risk assessment. In 2013, Martin and coworkers reported a new equation for estimating LDL-C that was more accurate than the Friedewald equation when compared to ultracentrifugation method (also known as β quantification). This new equation was reported to overcome the poor performance of the Friedewald equation at TG >150 mg/dL (>1.7 mmol/L) and at low concentrations of LDL-C (<70 mg/dL or <1.8 mmol/L), which may now be more common and clinically relevant owing to the use of more effective lipid-lowering therapy. Not all studies, however, have shown a significant difference between the old and new LDL-C equations when compared to preparative ultracentrifugation, the gold standard method for measuring LDL-C. Nevertheless, many clinical laboratories and at least one major provider of diagnostic testing services in the US switched to the new equation in 2017. In 2018, the American College of Cardiology and the American Heart Association (ACC/AHA) published new guidelines on the …

High density lipoproteins of blood plasma as a transport form of actinomycin D

Introduction.The development of new and highly effective antitumor therapy is one of the priorities of pharmacology. The paper presents one of the solutions to the problem related to the development of transport forms of antitumor drugs.The aimof the study was to study the ability of various fractions of plasma lipoproteins (VLDLP, LDL, HDL) to interact with actinomycin D and show the role of HDL as a transport form of actinomycin D in the body cells.Material and methods. The studies were conducted using unlabeled and tritium-labeled actinomycin D, preparative ultracentrifugation of the rat plasma lipoprotein fractions, chromatography, and in vivo experiments with intravenous administration of HDL complexes with labeled actinomycin D.Results.The important role of HDL in the formation of complexes with actinomycin D in comparison with LDL and LPA was shown. The basic physicochemical characteristics of the interaction of HDL and apolipoprotein A-I with actinomycin were obtained. The constants of the association were of the order of 105 M-1, and the number of binding sites for the drug was 26 for HDL and 12 for apolipoprotein A-I. In vivo studies on rats, the highest radioactivity after intravenous injection of HDL complexes with tritium-labelled actinomycin D was observed in the adrenal glands, then in the liver and kidneys. The uptake of tritium-labelled actinomycin D was twice lower in the lungs, adipose tissue, thymus and spleen. The low uptake of the label was observed in the myocardial tissue.Conclusion.The results obtained demonstrate the feasibility of using HDL as a transport form of actinomycin D in body cells.

Time to Make a Change: Assessing LDL-C Accurately in the Era of Modern Pharmacotherapeutics and Precision Medicine

The Friedewald equation for estimation of low-density lipoprotein cholesterol (LDL-C) was published in 1972 as an alternative to direct assessment by preparative ultracentrifugation. In this equation, very low-density lipoprotein is estimated by dividing triglycerides by a fixed factor (5 in mg/dL or 2.2 in mmol/L) and subtracting this term from non-high-density lipoprotein cholesterol (non-HDL-C). This method was derived in fasting samples from a small cohort of patients with primarily genetic dyslipidemias followed at the NIH. The method served well as the global standard for LDL-C estimation for decades, but is not well suited to modern clinical practice because it tends to underestimate LDL-C at low LDL-C and high triglyceride levels. The concern is that underestimation could lead to undertreatment in high-risk patients. Derived from big data and now validated around the world, a novel LDL-C equation created at Johns Hopkins replaces the fixed factor seen in the classic equation with a patient-specific variable based on triglyceride and non-HDL-C levels. Given its superior accuracy in fasting and non-fasting populations alike, the novel equation is now the preferred method for LDL-C estimation and is being incorporated by leading clinical laboratories.

DNA Content in Extracellular Vesicles Isolated from Porcine Coronary Venous Blood Directly after Myocardial Ischemic Preconditioning

BackgroundExtracellular vesicles (EV) are nano-sized membranous structures released from most cells. They have the capacity to carry bioactive molecules and gene expression signals between cells, thus mediating intercellular communication. It is believed that EV confer protection after ischemic preconditioning (IPC). We hypothesize that myocardial ischemic preconditioning will lead to rapid alteration of EV DNA content in EV collected from coronary venous effluent.Materials and MethodsIn a porcine myocardial ischemic preconditioning model, EV were isolated from coronary venous blood before and after IPC by differential centrifugation steps culminating in preparative ultracentrifugation combined with density gradient ultracentrifugation. The EV preparation was validated, the DNA was extracted and further characterized by DNA sequencing followed by bioinformatics analysis.ResultsPorcine genomic DNA fragments representing each chromosome, including mitochondrial DNA sequences, were detected in EV isolated before and after IPC. There was no difference detected in the number of sequenced gene fragments (reads) or in the genomic coverage of the sequenced DNA fragments in EV isolated before and after IPC. Gene ontology analysis showed an enrichment of genes coding for ion channels, enzymes and proteins for basal metabolism and vesicle biogenesis and specific cardiac proteins.ConclusionsThis study demonstrates that porcine EV isolated from coronary venous blood plasma contain fragments of DNA from the entire genome, including the mitochondria. In this model we did not find specific qualitative or quantitative changes of the DNA content in EV collected immediately after an in vivo myocardial IPC provocation. This does not rule out the possibility that EV DNA content changes in response to myocardial IPC which could occur in a later time frame.

Enrichment and characterization of ferritin for nanomaterial applications

Ferritin is a ubiquitous iron storage protein utilized as a nanomaterial for labeling biomolecules and nanoparticle construction. Commercially available preparations of horse spleen ferritin, widely used as a starting material, contain a distribution of ferritins with different iron loads. We describe a detailed approach to the enrichment of differentially loaded ferritin molecules by common biophysical techniques such as size exclusion chromatography and preparative ultracentrifugation, and characterize these preparations by dynamic light scattering, and analytical ultracentrifugation. We demonstrate a combination of methods to standardize an approach for determining the chemical load of nearly any particle, including nanoparticles and metal colloids. Purification and characterization of iron content in monodisperse ferritin species is particularly critical for several applications in nanomaterial science.

Characterization of Particle Size and Surface Adsorption for SiO2 Abrasives Used in Chemical Mechanical Planarization via Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is shown to be an effective characterization tool in the analysis of silica abrasives used in CMP slurries. Tagging of silica particles via non-covalent adsorption of a highly fluorescent dye is the key step in applying the technique to these materials. FCS is shown to have ample analytical sensitivity to detect and analyze the smallest fraction (< 20 nm) of an abrasive silica dispersion. Fractionation of abrasive silica dispersions using preparative ultracentrifugation allows a direct comparison of FCS and dynamic light scattering (DLS) methods for sizing particles with diameters less than 20 nm in the dispersion. The sensitivity of FCS measurements of this type exceeded the sensitivity of DLS determinations in this size range. Non-covalent adsorption of a fluorescent dye by the silica particle enables the characterization of adsorption behaviors of CMP slurry additives, such as benzotriazole (BTA) and amino acids, on abrasive silica particles. Different adsorption behaviors are observed and the use of one specific fluorescent dye, Rhodamine 110, afforded a determination of a quantitative isotherm for dye adsorption. Future applications of the FCS method for sizing abrasive nano-particles and adsorption isotherm analysis of other CMP abrasives, such as alumina and ceria, are proposed.

Large scale purification of RNA nanoparticles by preparative ultracentrifugation.

Purification of large quantities of supramolecular RNA complexes is of paramount importance due to the large quantities of RNA needed and the purity requirements for in vitro and in vivo assays. Purification is generally carried out by liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE), or agarose gel electrophoresis (AGE). Here, we describe an efficient method for the large-scale purification of RNA prepared by in vitro transcription using T7 RNA polymerase by cesium chloride (CsCl) equilibrium density gradient ultracentrifugation and the large-scale purification of RNA nanoparticles by sucrose gradient rate-zonal ultracentrifugation or cushioned sucrose gradient rate-zonal ultracentrifugation.

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Objectives Preeclampsia (PE) and IUGR share common pathomechanisms. Recently, we reported on serum lipid concentrations in both conditions in the early-onset form of PE before 34 weeks of gestation (Pecks et al., 2012). Subtle changes of triglyceride concentrations were noticed in PE while a significant decrease in LDL levels was found in IUGR subjects when compared to a control group (CTRL). We now aimed to extend our lipid analysis within our cohort to gain a deeper understanding of lipid metabolism and its role in the pathogenesis of both diseases. Methods Serum of (i) PE, (ii) PE with IUGR, and (iii) normotensive IUGR each n = 10 was matched to a CTRL group (iv, n = 30). HDL, IDL, LDL, VLDL were isolated by sequential preparative ultracentrifugation. Esterified and non-esterified cholesterol, phospholipid, and triglyceride concentrations were determined in each fraction. Statistical analysis was done using 2-way ANOVA. Results No significant difference between the case groups and CTRL could be observed in the HDL fraction. In the IDL fraction triglyceride levels were higher in PE as compared to CTRL. Within the LDL fraction esterified and non-esterified cholesterol and phospholipid levels were lower in IUGR and IUGR + PE. Within the VLDL fraction triglycerides were higher in all three case groups as compared to CTRL (table). Conclusions Our results suggest a disturbed conversion of VLDL to LDL is involved in both IUGR and preeclampsia. Triglyceride metabolism appears to be most affected in preeclampsia whereas cholesterol metabolism is disturbed in IUGR irrespectively of additional preeclampsia. One has to carefully separate all three entities to avoid misinterpretation. Disclosures U. Pecks: None. B. Winkler: None. T. Goecke: None. N. Maass: None. C. Contini: None. K. Winkler: None.

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Effects of Evolocumab (AMG 145), a Monoclonal Antibody to PCSK9, in Hypercholesterolemic, Statin-Treated Japanese Patients at High Cardiovascular Risk

YUKAWA is a 12-week, randomized, double-blind, placebo-controlled, phase 2 study evaluating the efficacy and safety of evolocumab (AMG 145) in statin-treated Japanese patients at high cardiovascular risk. 310 eligible patients receiving stable statin (±ezetimibe) therapy were randomized to 1 of 6 treatments: placebo every 2 weeks (Q2W) or monthly (QM), evolocumab 70 mg or 140 mg Q2W, or evolocumab 280 mg or 420 mg QM. The primary endpoint was the percentage change from baseline in low-density lipoprotein cholesterol (LDL-C) measured by preparative ultracentrifugation (UC). Secondary endpoints included percentage changes in other lipid parameters and the proportion of patients with LDL-C <1.8 mmol/L. Mean (SD) age was 62 (10) years; 37% were female; and the mean (SD) baseline LDL-C was 3.7 (0.5) mmol/L (by UC). Mean (SE) changes vs. placebo in LDL-C were greatest in the high-dose groups: -68.6 (3.0) % and -63.9 (3.2) % with 140 mg Q2W and 420 mg QM dosing, respectively. Up to 96% of evolocumab-treated patients achieved LDL-C <1.8 mmol/L. Adverse events (AEs) were more frequent in evolocumab (51%) vs. placebo (38%) patients; 4 patients taking evolocumab discontinued treatment because of an AE. There were no significant differences in AE rates based on dose or dose frequency. In Japanese patients at high cardiovascular risk with hypercholesterolemia on stable statin therapy, evolocumab significantly reduced LDL-C and was well tolerated during this 12-week study.

A CHO/fibre diet reduces and a MUFA diet increases postprandial lipaemia in type 2 diabetes: no supplementary effects of low-volume physical training

The aim of the study was to evaluate the effects of a supervised physical training added to a healthy diet-rich in either carbohydrate and fibre (CHO/fibre) or monounsaturated fatty acids (MUFA)-on postprandial dyslipidaemia, an independent cardiovascular risk factor particularly relevant in type 2 diabetes (T2D). Participants were forty-five overweight/obese subjects with T2D, of both genders, in good blood glucose control with diet or diet+metformin, with normal fasting plasma lipids. According to a parallel groups 2×2 factorial design, participants were randomized to an 8-week isoenergetic intervention with a CHO/fibre or a MUFA diet, with or without a supervised low-volume aerobic training programme. The main outcome of the study was the incremental area under the curve (iAUC) of lipid concentrations in the plasma chylomicron+VLDL lipoprotein fraction, isolated by preparative ultracentrifugation (NCT01025856). Body weight remained stable during the trial in all groups. Physical fitness slightly improved with training (VO2 peak, 16±4 vs. 15±3ml/kg/min, M±SD, p<0.05). Postprandial triglyceride and cholesterol iAUCs in plasma and chylomicron+VLDL fraction decreased after the CHO/fibre diet, but increased after the MUFA diet with a significant effect for diet by two-way ANOVA (p<0.05). The addition of exercise training to either dietary intervention did not significantly influence postprandial lipid response. A diet rich in carbohydrates and fibre reduced postprandial triglyceride-rich lipoproteins compared with a diet rich in MUFA in patients with T2D. A supervised low-volume physical training did not significantly influence these dietary effects.

Formation Kinetics and Structural Features of Beta‐Amyloid Aggregates by Sedimented Solute NMR

The accumulation of soluble toxic beta-amyloid (Aβ) aggregates is an attractive hypothesis for the role of this peptide in the pathology of Alzheimer's disease. We have introduced sedimentation through ultracentrifugation, either by magic angle spinning (in situ) or preparative ultracentrifuge (ex situ), to immobilize biomolecules and make them amenable for solid-state NMR studies (SedNMR). In situ SedNMR is used here to address the kinetics of formation of soluble Aβ assemblies by monitoring the disappearance of the monomer and the appearance of the oligomers simultaneously. Ex situ SedNMR allows us to select different oligomeric species and to reveal atomic-level structural features of soluble Aβ assemblies.

Assessment and significance of protein-protein interactions during development of protein biopharmaceuticals.

Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibody-antigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.