A simple fluorescent method for simultaneous determination of aortic permeability to horseradish peroxidase and bovine serum albumin.

Differences in regional aortic net uptake of bovine serum albumin (BSA) and horseradish peroxidase (HP) have been examined by means of conjugation of these molecules to the fluorescent protein tracers fluorescein isothiocyanate (FITC) and lissamine rhodamine B (RB200). Using male Wistar rats, uptake of FITC-BSA under steady state conditions in the ascending aorta and aortic arch (14 +/- 2 micrograms/mg aorta) is significantly higher (p less than 0.05) than that of either the upper, middle, or lower third of the thoracic aorta (10 +/- 1 mu, 9 +/- 1 mu, and 8 +/- 1 micrograms/mg, respectively). No regional variation in net uptake of RB200-HP was observed in these same aortic regions, with respective mean values (+/- SE) being 69 +/- 2, 69 +/- 2, 68 +/- 4, and 68 +/- 4 micrograms/mg. Examination of fluorescence photomicrographs indicate that FITC-BSA is localized along the collagen-elastin bands, while the RB200-HP is found between these bands. Differences between FITC-BSA and RB200-HP uptake and deposition reflect such things as differences in uptake rates as influenced by initial concentrations, permeability coefficients, as well as possible differences in molecular charge and affinity. The results indicate that the fluorometric procedures described in this investigation are simple, sensitive, and quantitative, and suitable for simultaneous measurement of aortic uptake of molecules having different molecular sizes as well as for the intraaortic localization of these substances.