Introduction: A major goal of the 21st century is to mitigate disease and healthcare costs. Coronary artery disease (CAD) kills 370,000 Americans and costs the United States $109 billion each year. More accurate screening tools are needed to ameliorate CAD related morbidity and mortality. A new approach to the direct analysis of blood serum led to the development of a prospective clinical screening tool for CAD involving sodium dodecyl sulfate (SDS) capillary gel electrophoresis (CGE). Hypothesis: The novel method generates serum protein profiles which can be used to screen individuals for heart disease by distinguishing profile features of macromolecules involved in CAD. Methods: Serum profiles were generated by SDS-CGE for each study participant (n=48), 30 with CAD and 18 ‘NonCAD’ as identified by histories of CAD or unremarkable coronary angiographies. Univariate and multivariate analyses of the profile features were conducted to identify biomarkers and statistically classify participants as CAD or NonCAD. To compare the accuracy of the method with that of an established clinical tool, participants’ 10 Year Framingham Risk Scores (FRS) were calculated. Individuals with FRS scores less than 10% (low) risk were classified as NonCAD and those with greater than 10% (moderate to severe) risk were classified as CAD. Results: A “serum protein fingerprint” profile is shown. Relative standard deviations of 2% for MW and 20% for absorbance indicate method repeatability. In the classification of participants as CAD or NonCAD, the respective accuracies of the multivariate canonical and linear discriminant analyses were 90% (p-value of 7E-5) and 94%. The classification accuracy of FRS was less than 42%. Two features of the serum profiles (#’s 3 and 7 (with Prob>F values of 0.01 and 4E-5 respectively)) are potentially novel markers for CAD. Conclusions: The proposed serum protein analysis method correlated proteins with CAD and was more accurate than the FRS method in this study.

The effect of α-linolenic acid from a flaxseed (FLX)-enriched diet on plasma lipid and fatty acid metabolism and possible atherosclerosis risk factors was studied in Monk parrots (Myiopsitta monachus). Twenty-four Monk parrots were randomly assigned to diets containing either 10% ground SUNs or 10% ground FLXs. Feed intake was calculated daily. Blood samples, body condition scores and body weights were obtained at -5 weeks, day 0, 7, 14, 28, 42 and 70. Plasma samples were analysed for total cholesterol, free cholesterol, triacylglycerols and lipoproteins. Phospholipid subfraction fatty acid profiles were determined. By day 70, the FLX group had significantly higher plasma phospholipid fatty acids including 18:3n-3 (α-linolenic acid), 20:5n-3 (eicosapentaenoic acid) and 22:6n-3 (docosahexaenoic acid). The sunflower group had significantly higher plasma phospholipid levels of 20:4n-6 (arachidonic acid). By day 70, the high-density lipoprotein (HDL) peak shifted resulting in significantly different HDL peak densities between the two experimental groups (1.097 g/ml FLX group and 1.095 g/ml SUN group, p = 0.028). The plasma fatty acid results indicate that Monk parrots can readily convert α-linolenic acid to the long-chain omega-3 derivatives including docosahexaenoic acid and reduce 20:4n-6 accumulation in plasma phospholipids. The reason for a shift in the HDL peak density is unknown at this time.

To evaluate potential factors related to avian atherosclerosis, plasma cholesterol and triglyceride values were measured in 35 apparently healthy captive monk parakeets (Myiopsitta monachus). Birds were categorized as healthy or at risk based on body condition score and weight and were also evaluated based on their aviary environmental conditions. Plasma cholesterol mean was 8.008mmol/L (range: 4.655 to 20.33mmol/L) or 309.65mg/dl (range: 180 to 786mg/dl) for all birds sampled. Plasma triglyceride mean for all birds sampled was 4.364mmol/L (range: 0.960 to 44.62mmol/L) or 386.54mg/dl (range: 85 to 3952mg/dl). Thirty plasma samples were evaluated through density gradient ultracentrifugation lipid profiling techniques used to examine risk of cardiovascular disease in humans. The resultant lipid density profile graph was determined from the hydrated densities of the following lipids: triglyceride-rich lipoproteins, low-density lipoproteins (LDL) and subfractions, and high-density lipoproteins and subfractions. When analyzed using linear discriminant analysis, lipid profiles of triglyceride-rich lipoproteins, LDL1, LDL2, and high-density lipoprotein 2b subfractions were increased (P < 0.05) in at-risk monk parakeets when compared with healthy cohorts. Gender and diet had no apparent effect on plasma cholesterol or triglyceride concentrations. Cholesterol and triglyceride concentrations and lipoprotein density profiles from captive monk parakeets, a species known to be affected by atherosclerosis, may prove useful as markers for use in future investigation of atherosclerosis in birds. However, the consequence of increased plasma lipid concentrations and changes of lipoprotein profiles on avian health requires additional investigation.

BACKGROUND:Despite the importance of abnormalities in lipoprotein metabolism in clinical canine medicine, the fact that most previously used methods for lipoprotein profiling are rather laborious and time-consuming has been a major obstacle to the wide clinical application and use of lipoprotein profiling in this species. The aim of the present study was to assess the feasibility of a continuous lipoprotein density profile (CLPDP) generated within a bismuth sodium ethylenediaminetetraacetic acid (NaBiEDTA) density gradient to characterize and compare the lipoprotein profiles of healthy dogs of various breeds, healthy Miniature Schnauzers, and Miniature Schnauzers with primary hypertriacylglycerolemia. A total of 35 healthy dogs of various breeds with serum triacylglycerol (TAG) and cholesterol concentrations within their respective reference intervals were selected for use as a reference population. Thirty-one Miniature Schnauzers with serum TAG and cholesterol concentrations within their respective reference intervals and 31 Miniature Schnauzers with hypertriacylglyceridemia were also included in the study.RESULTS:The results suggest that CLPDP using NaBiEDTA provides unique diagnostic information in addition to measurements of serum TAG and cholesterol concentrations and that it is a useful screening method for dogs with suspected lipoprotein metabolism disorders. Using the detailed and continuous density distribution information provided by the CLPDP, important differences in lipoprotein profiles can be detected even among dogs that have serum TAG and cholesterol concentrations within the reference interval. Miniature Schnauzers with serum TAG and cholesterol concentrations within the reference interval had significantly different lipoprotein profiles than dogs of various other breeds. In addition, it was further established that specific lipoprotein fractions are associated with hypertriacylglyceridemia in Miniature Schnauzers.CONCLUSIONS:The results of the present study suggest that density gradient ultracentrifugation using NaBiEDTA is a useful screening method for the study of lipoprotein profiles in dogs. Therefore, this method could potentially be used for diagnostic purposes for the separation of dogs suspected of having lipoprotein abnormalities from healthy dogs.

Summary In many oviparous animals, bursting type atresia of ovarian follicles occurs during the reproductive cycle resulting in the escape of yolk into the extracellular compartment. In birds, this ectopic yolk is rapidly cleared by an unknown process that involves the appearance of yolk-engorged macrophage-like cells. To study this unique type of lipid transport, we injected young male chickens intra-abdominally with egg yolk. Absorption of egg yolk from the body cavity markedly increased the triacylglyceride-rich fraction (TRL) of plasma lipoproteins and was coincident with increased levels of plasma triacylglycerides (TAG) but not non-esterified fatty acids (NEFA). Thus, the transport of yolk lipids from the abdominal cavity appears to occur in lipoproteins and be more similar to the transport of hepatic TAG to the periphery via lipoproteins than to transport of adipose TAG to the periphery via NEFA released by the action of lipases. When macrophages were exposed to yolk in vitro, they quickly phagocytized yolk; however, it is unclear if this level of phagocytosis contributes significantly to total yolk clearance. Instead, the chicken macrophage may function more as a facilitator of yolk clearance through modification of yolk lipoproteins and the regulation of the local and systemic immune response to ectopic yolk. Yolk appears to be anti-inflammatory in nature. Yolk did not increase inflammatory cytokines IL-1, IL-6 and IFNγ either in vivo or in vitro; in fact, yolk dampened many inflammatory changes caused by lipopolysaccharide (LPS). Conversely, LPS-induced inflammation retarded yolk clearance from the abdominal cavity and plasma TAG levels.

AIMS:We discovered that some adults with coronary heart disease (CHD) have a high density lipoprotein (HDL) subclass which induces human aortic smooth muscle cell (ASMC) apoptosis in vitro. The purpose of this investigation was to determine what properties differentiate apoptotic and non-apoptotic HDL subclasses in adults with and without CHD.METHODS AND RESULTS:Density gradient ultracentrifugation was used to measure the particle density distribution and to isolate two HDL subclass fractions, HDL2 and HDL3, from 21 individuals, including 12 without CHD. The HDL fractions were incubated with ASMCs for 24 h; apoptosis was quantitated relative to C2-ceramide and tumour necrosis factor-alpha (TNF-α). The observed effect of some HDL subclasses on apoptosis was ∼6-fold greater than TNF-α and ∼16-fold greater than the cell medium. We observed that apoptotic HDL was (i) predominately associated with the HDL2 subclass; (ii) almost exclusively found in individuals with a higher apoC-I serum level and a novel, higher molecular weight isoform of apoC-I; and (iii) more common in adults with CHD, the majority of whom had high (>60 mg/dL) HDL-C levels.CONCLUSIONS:Some HDL subclasses enriched in a novel isoform of apoC-I induce extensive ASMC apoptosis in vitro. Individuals with this apoptotic HDL phenotype generally have higher apoC-I and HDL-C levels consistent with an inhibitory effect of apoC-I on cholesteryl ester transfer protein activity. The association of this phenotype with processes that can promote plaque rupture may explain a source of CHD risk not accounted for by the classical risk factors.

A prior study showed that students best make predictions about the outcome of opening a valve between two flasks containing a fluid or vacuum when they view both a demonstration video and a particulate animation, but the study showed no influence from the order in which these visualizations were used. The purpose of this current research was to study the effect of the order of visualization on students' particulate-level explanations. For this study, first-year general chemistry students in a south-central university in the United States were asked to choose, or type in their own explanations, for three experiments involving diffusion effusion. Student ability to focus on particulate explanations was investigated after viewing either a macroscopic demonstration or an animated particle view. Students were directed to a Web site where they received one of two randomly assigned treatments. One group of students was shown the particulate animation first, followed by the macroscopic demonstration. For the other group, the order was reversed. Student explanations were assessed after each view. Finally, both groups were shown a side-by-side view of the animation and demonstration and their explanation was assessed one final time. Results showed that the order of visualizations did make a difference, with the macroscopic view followed by the particle view yielding significantly more particulate explanations.

Early detection of the beginning stage of cardiovascular disease (CVD) is an approach to prevention because the process is reversible at this stage. Consequently, several methods for screening for CVD have been introduced in recent years incorporating different analytical methods for characterizing the population of blood-borne lipoprotein subclasses. The gold standard method for lipoprotein subclassification is based on lipoprotein density measured by sedimentation equilibrium using the ultracentrifuge. However, this method has not been adopted for clinical studies because of difficulties in achieving the precision required for distinguishing individuals with and without CVD particularly when statistical classification methods are used. The objective of this study was to identify and improve the major factors that influence the precision of measurement of lipoprotein density profile by sedimentation equilibrium analysis and labeling with a fluorescent probe. The study has two phases, each contributing to precision. The first phase focuses on the ultracentrifugation-related variables, and the second phase addresses those factors involved in converting the fluorescent lipoprotein density profile to a digital format compatible with statistical analysis. The overall improvement in precision was on the order of a factor of 5, sufficient to be effectively applied to ongoing classification studies relating to CVD risk assessment.

Apolipoprotein C-I (apoC-I) is a 6.6 kDa serum protein associated with high density lipoproteins (HDL) and triglyceride-rich lipoproteins. In this study, apoC-I was examined in high density lipoprotein subfractions from individuals with and without coronary artery disease (CAD). New isoforms of apoC-I, were detected in the cohort of individuals with CAD using mass spectrometry while the expected apoC-I isoforms were absent. In addition, the apoC-I mass spectra for the CAD cohort had satellite peaks indicative of the involvement of oxidative processes. Further analysis of the mass spectra of the CAD and non-CAD cohorts suggest that the origin of these new isoforms may be due to genetic mutations that could compromise the function of apoC-I.

Although HDL-mediated cholesterol transport to the liver is well studied, cholesterol efflux from hepatocytes back to HDL is less well understood. Real-time imaging of efflux of 22-( N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), which is poorly esterified, and [ 3 H]cholesterol, which is extensively esterified, from cultured primary hepatocytes of wild-type and sterol carrier protein-2 (SCP-2) gene-ablated mice showed that 1) NBD-cholesterol efflux was affected by the type of lipoprotein acceptor, i.e., HDL3 over HDL2; 2) NBD-cholesterol efflux was rapid (detected in 1–2 min) and resolved into fast [half time ( t ½ ) = 2.4 min, 6% of total] and slow ( t ½ = 26.5 min, 94% of total) pools, consistent with protein- and vesicle-mediated cholesterol transfer, respectively; 3) SCP-2 gene ablation increased efflux of NBD-cholesterol, as well as [ 3 H]cholesterol, albeit less so due to competition by esterification of [ 3 H]cholesterol, but not NBD-cholesterol; and 4) SCP-2 gene ablation increased initial rate (2.3-fold) and size (9.7-fold) of rapid effluxing sterol, suggesting an increased contribution of molecular cholesterol transfer. In addition, colocalization, double-immunolabeling fluorescence resonance energy transfer, and electron microscopy, as well as cross-linking coimmunoprecipitation, indicated that SCP-2 directly interacted with the HDL receptor, scavenger receptor class B type 1 (SRB1), in hepatocytes. Other membrane proteins in cholesterol efflux [SRB1 and ATP-binding cassettes (ABC) A-1, ABCG-1, ABCG-5, and ABCG-8] and several soluble/vesicle-associated proteins facilitating intracellular cholesterol trafficking (StARDs, NPCs, ORPs) were not upregulated. However, loss of SCP-2 elicited twofold upregulation of liver fatty acid-binding protein (L-FABP), a protein with lower affinity for cholesterol but higher cytosolic concentration than SCP-2. Ablation of SCP-2 and L-FABP decreased HDL-mediated NBD-cholesterol efflux. These results indicate that SCP-2 expression plays a significant role in HDL-mediated cholesterol efflux by regulating the size of rapid vs. slow cholesterol efflux pools and/or eliciting concomitant upregulation of L-FABP in cultured primary hepatocytes.

Synopsis: We have identified a high-density lipoprotein (HDL) phenotype in a cohort of adult patients with coronary heart disease and hyperalphalipoproteinemia (HALP) that appears to promote atherosclerosis on the basis of its in vitro properties. Purpose: The purpose of this study is to characterize the composition and functional properties of this HDL by the use of a novel approach for lipoprotein characterization and isolation coupled with series of in vitro experiments aimed at elucidating the atherogenic effects on vascular cells. Methods: This atherogenic HDL subclass was characterized and isolated by the use of an innovative approach based on density gradient ultracentrifugation (DGU) and a variant of this method, immunospecific DGU. We also introduce a novel statistical classification method for studying the association of lipoprotein subclasses with coronary heart disease (CHD) risk on the basis of the synergistic, circulating distribution of all lipoprotein subclass particles rather than the association with a specific subclass or lipid level. The combination of these two approaches is used to help us distinguish individuals with high HDL-C levels who may have an atherogenic form of HDL. To validate the atherogenic functional properties of this HDL subclass, the HDL2 and HDL3 subclasses were incubated with cultured human aortic smooth muscle (HASMC) and endothelial cells (HAEC) in a series of experiments aimed at identifying the molecular mechanisms that might promote atherosclerosis. Results: Compositional analyses reveal that the buoyant HDL2 subclass is enriched in apoC-I compared to Compositional analyses reveal that the buoyant HDL2 subclass is enriched in apoC-I compared with the HDL3 subclass in subjects with HALP and CHD. This HDL2 fraction induced striking apotosis (∼50%) in cultured HASMC. Such apotosis was markedly reduced when cells were incubated with apoC-I−depleted lipoproteins and when cells were preincubated with GW4869, an inhibitor of N-SMase. When HAEC were incubated with the same HDL2 fractions, we observed massive nitric oxide production and increased intracellular adhesion molecule-1 expression. Conclusions: apoC-I−enriched HDL is a sinister risk factor that acts like a Trojan horse: Subjects have high HDL-C levels and are perceived to have protection from CHD, whereas at the vascular level, this HDL subclass causes endothelial cell inflammation increasing the risk of plaque formation with eventual destabilization associated with smooth muscle cell apotosis.

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/ cholesteryl esters) and males (cholesterol/ cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDLreceptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/ or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3 alpha-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/ 8, and apolipoprotein A1), or oxidation/ transport of bile salts (cholesterol 7 alpha-hydroxylase, sterol 27 alpha-hydroxylase, Na+/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/ transport (viaOapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpressionmice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.-Atshaves, B. P., A. L. McIntosh, G. G. Martin, D. Landrock, H. R. Payne, S. Bhuvanendran, K. K. Landrock, O. I. Lyuksyutova, J. D. Johnson, R. D. Macfarlane, A. B. Kier, and F. Schroeder. Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice. J. Lipid Res. 2009. 50: 1429-1447.