Size and conformation of Ficoll as determined by size-exclusion chromatography followed by multiangle light scattering

摘要

The characteristics of the glomerular filtration barrier (GFB) are challenging to measure, as macromolecular solutes in blood may be metabolized or transported by various cells in the kidney. Urinary solute concentrations generally reflect the cumulative influence of multiple transport processes rather than the intrinsic behavior of the GFB alone. Synthetic tracer molecules which are not secreted, absorbed, or modified by the kidney are useful tools. Ficoll, a globular polymer of epichlorohydrin and sucrose, is round, physiologically inert, and easily labeled, making it a nearly ideal glomerular probe. Fissell et al. reported filtration data suggesting that Ficoll was not as spherical as had been previously suggested (Fissell WH, Manley S, Dubnisheva A, Glass J, Magistrelli J, Eldridge AN, Fleischman AJ, Zydney AL, Roy S. Am J Physiol Renal Physiol 293: F1209–F1213, 2007). More recently, two investigators published comparisons of neutral and anionic Ficoll clearance that suggest Ficoll may undergo conformational changes when chemically derivatized (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083–F1089, 2006; Guimaraes MAM, Nikolovski J, Pratt LM, Greive K, Comper WD. Am J Physiol Renal Physiol 285: F1118–F1124, 2003). To investigate Ficoll's characteristics further, we examined two commercial preparations, Ficoll 70 and Ficoll 400, by size-exclusion chromatography using a differential refractive index detector combined with light-scattering and viscosity detectors. A slope of 0.45 was obtained from the plot of the logarithm of molecular mass against the logarithm of root-mean square radius. The Mark-Houwink exponent values of 0.34 and 0.36 were calculated for Ficoll 70 and Ficoll 400, respectively. These results suggest Ficoll's conformation in physiological saline solution is likely intermediate between a solid sphere and a well-solvated linear random coil. The measurements help explain our previous observations and guide interpretation of in vivo experiments.