Quantitative sphingosine measurement as a surrogate for total ceramide concentration-preclinical and potential translational applications.

Biomarkers are an increasingly important constituent of the drug development process, offering the potential of increased efficiency through reduced compound attrition and earlier proof of mechanism and/or efficacy. Assays developed for compound screening that can be directly translated for clinical trials are especially valuable, but their successful adoption requires a careful balance between assay performance and implementation costs. One such 'fit-for-purpose' biomarker assay, the indirect measurement of pharmacological modulation of sphingolipid biosynthesis and disposition, is presented here. Among sphingolipids, numerous ceramide species are readily detectable in different lipoprotein fractions of mammalian plasma, but their parallel quantification can be prohibitively expensive and time consuming. Ceramides differ in their fatty acid moiety, which is readily removed by hydrolysis, yielding a common sphingosine derivative, the measurement of which serves as an indicator of total ceramide. When followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) for detection, robust analyte quantification becomes relatively straightforward. The practical utility of a method developed to be fit for the purpose of rapidly and quantitatively measuring treatment-induced variations in total ceramide from hamster plasma and individual lipoprotein fractions is described. With a linear calibration range from 0.003 to 33.4 microm sphingosine, precision and accuracy error in plasma-based quality controls spiked with ceramides was less than 15%. The specificity of the assay for ceramides was also assessed. The simplicity of the method would allow for its potential translation to other preclinical species, as well as for clinical applications in later-stage drug development.

Isoform-specific interactions of human apolipoprotein E to an intermediate conformation of human Alzheimer amyloid-beta peptide.

Brain plaque deposits of amyloid-beta peptide (Abeta) is a pathological hallmark of Alzheimer's disease (AD) and apolipoprotein E (apoE) is thought to be involved in its deposition. One hypothesis for the role of apoE in the pathogenesis of AD is that apoE may be involved in deposition or clearance of Abeta by direct protein-to-protein interaction. Lipidated apoE4 bound preferentially to an intermediate aggregated form of Abeta and formed two- to three-fold more binding complexes than isoforms apoE2 or apoE3. The interaction was detected by a sandwich ELISA with capture antibodies specific for the N-terminus of apoE, whereas the interaction was not recognized with a C-terminal antibody. The observations indicate that the C-terminus of apoE4 interacts with the intermediate form of Abeta. The differential risk of AD related to apoE genotype may be the result of an enhanced capacity of apoE4 binding to an intermediate aggregated form of Abeta.

Atherosclerosis in Octodon degus (degu) as a model for human disease.

OBJECTIVE: Animal models of atherosclerosis are essential to elucidate disease mechanisms and develop new therapies. Each model features advantages and disadvantages in exemplifying the pathophysiology of human atherosclerosis. Diet-induced development of atherosclerosis in Octodon degus (degu) was examined to demonstrate the potential of the degu as a model of human atherosclerosis. METHODS: Degus were fed for 16 weeks with either normal chow or chow containing 0.25% cholesterol and 6% palm oil to induce atherosclerosis. The lipid compositions of plasma lipoproteins and aortas were determined. Locations of aortic lesions were mapped by imaging of fluorescently stained aortic lesions. Lesion morphology in the brachiocephalic artery was detected by histological staining. RESULTS: Total plasma cholesterol in chow-fed degus was distributed approximately 60% in HDL, 30% in LDL and less than 10% in VLDL. Cholesterol-fed degus exhibited 4- to 5-fold increases in total plasma cholesterol, principally in the VLDL and LDL fractions. Cholesteryl ester transfer protein activity of similar magnitude to that in human plasma was detected in chow-fed degu plasma. Cholesterol-fed degus developed cholesteryl ester-rich atherosclerotic lesions throughout the aorta. Histological examination of lesions in the brachiocephalic artery showed well-formed, foam cell-rich lesions populated with inflammatory cells. It is also noteworthy that all the degus in this study exhibited hyperglycemia. CONCLUSION: These results demonstrate that degus have a human-like lipoprotein metabolism and develop extensive atherosclerosis with cholesterol feeding in the presence of hyperglycemia. These features, combined with the manageable size and handling characteristics, point to the potential of the degu as a useful model for atherosclerosis research.