Colorimetric assay to quantify macromolecule diffusion across endothelial monolayers

Endothelial "capillary leak", the loss of vascular integrity in response to noxious stimuli, is characterized by extravasation of protein-rich fluid f rom capillary lumen into surrounding tissue interstitium. This increase in vascular permeability, in response to inflammatory mediators, correlates wi th endothelial cell contraction and the formation of intercellular gaps wit hin the monolayer. However, in vivo assessment of paracellular solute flow between endothelial cells may be complicated by multiple uncontrolled param eters. In vitro examinations of endothelial barrier leak have relied on ele ctrical impedence or macromolecule diffusion techniques to determine the de tails pertinent to capillary barrier function. In this report, a simple, se nsitive, nonradioactive, colorimetric assay to quantify the leak of a label ed protein marker across endothelial monolayers is described. This procedur e avoids the hazards of radioisotope labels and the technical limitations o f electrical resistance technology.