Preparation and high-performance size-exclusion chromatographic (HPSEC) analysis of fluorescein isothiocyanate-hydroxyethyl starch: macromolecular probes of the blood-lymph barrier.

A procedure is described for the preparation of fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES). Chromatographic techniques for the purification and analysis of FITC-HES that include the development of a high-performance size-exclusion chromatographic (HPSEC) technique with a fluorescence spectrophotometer-computer detection system are described. FITC-HES macromolecules have a wide range of hydrodynamic radii (ae greater than 120- less than 10 A) and the substitution ratio of FITC to the size-selected HES macromolecules remains constant throughout the chromatographic range. The predominant isoelectric point (pI) of the multiple acidic isomers of FITC-HES is 4.6. In vitro, the very large FITC-HES macromolecules (greater than 100 A) are rapidly degraded (15 sec) by alpha-amylase in control dog plasma. While most of the large molecules (100-20 A) remain intact for greater than 24 hr, this degradation is not associated with the loss of FITC from HES. In vivo, the rate of this reaction appeared to be accelerated and the degradation of FITC-HES between 0.1 and 12 hr was small. Illustration of the HPSEC quantitation of the spectrum of FITC-HES macromolecules in "near steady-state" lymph (L) and plasma (P) samples and the L/P ratio show that this technique can be used to describe the size selectivity of the blood-lymph barrier under conditions of unaltered capillary pressure. We propose that the size-selected solvent-drag reflection coefficient (sigma f) curves for the anionic FITC-HES under conditions of elevated capillary pressure is a measure of macromolecular convective permeability of the blood-lymph barrier.