Some Lessons Learned From a Comparison Between Sedimentation Velocity Analytical Ultracentrifugation and Size Exclusion Chromatography to Characterize and Quantify Protein Aggregates.

There are numerous problems with size exclusion chromatography (SEC), which often lead to inaccuracies in protein aggregate characterization. Hence, this study tested sedimentation velocity analytical ultracentrifugation (SV-AUC) as an orthogonal tool to SEC for quantifying the monomer and aggregates in intravenous immunoglobulin (IVIg) formulations. IVIg samples were subjected to agitation stress and analyzed using SEC mobile phases composed of 200 mM sodium phosphate (pH 7.0) with 0, 50, 100, 200, or 400 mM of NaCl. Surprisingly, 400 mM of NaCl was required in the mobile phase to attain complete protein recovery from the SEC column. Significant discrepancies between SEC and SV-AUC were reported when SEC analysis was performed using suboptimal concentrations (e.g., 0, 50, 100, and 200 mM) of NaCl in the mobile phase. The continuous sedimentation coefficient distributions obtained with SV-AUC resolved the high molecular weight species, whereas with SEC the high molecular weight species eluted as a single peak. Only with the orthogonal use of SV-AUC, we were able to develop a robust SEC method for accurate quantitation of monomer and aggregates in unagitated and agitated IVIg samples. Additionally, this article describes a modification to an existing method of quantitating insoluble aggregates from SV-AUC boundary data.

Regulation of vascular endothelial function by procyanidin-rich foods and beverages.

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response. Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC(50) values of 5.4, 1.6, 0.9, and 0.7 microM, respectively. Levels of ET-1 mRNA showed a similar pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.