ENZYMATIC TRACERS IN THE STUDY OF VASCULAR-PERMEABILITY

Elucidation of the ultrastructural basis of vascular permeability was aided by the development of cytochemical techniques for visualizing the distribution, within the vessel wall, of intravenously injected peroxidatic enzymes of varying molecular size. Tracer enzymes available range from 10 Å (hemeoctapeptide) to 52 Å (catalase) effective molecular radius. The use of enzymatic probe molecules assumes a thorough characterization of: (a) the molecular charge (isoelectric point of the native enzyme, and when feasible, its polyanionic and polycationic derivatives; (b) effective molecular radius (a(e)); (c) peroxidase activity (to detect by spectrophotometry of DAB-oxidizing activity, the optimal pH, temperature, and enzyme concentration to be employed in the cytochemical procedure). Molecular shape and state of dispersion of the enzymatic probes should be determined by gel chromatography and spectrophotometry of both the tracer solution and aliquots of blood plasma collected after i.v. injection of the tracer. Conditions required for the probe administration include: (a) the investigation of potential side effects (tests for toxicity and vascular leakage) and (b) estimation of the tracer volume and concentration which does not affect significantly the blood volume and osmotic pressure. Determination in vitro of the crosslinking of tracer molecules induced by the aldehyde fixative to be employed, also gives an indication on potential diffusion artifacts. Based on the formation thus obtained, the design of the cytochemical procedure should also take into account the possible use of methods for enhancing the peroxidatic reaction product: nitrogenous ligands (imidazole, diaminopyrimidine, histidine) or polyphenolic mordants (galloylglucoses). The usefulness of peroxidatic tracers in the investigation of vascular permeability is exemplified by some results obtained on the microvascular endothelium in vivo (transcytosis, intercellular pathway, etc.) and on endothelial cells isolated from heart microvasculature.